WO2022154119A1 - Method for processing soluble gpc3-containing specimens in soluble gpc3 immunoassays - Google Patents
Method for processing soluble gpc3-containing specimens in soluble gpc3 immunoassays Download PDFInfo
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- WO2022154119A1 WO2022154119A1 PCT/JP2022/001385 JP2022001385W WO2022154119A1 WO 2022154119 A1 WO2022154119 A1 WO 2022154119A1 JP 2022001385 W JP2022001385 W JP 2022001385W WO 2022154119 A1 WO2022154119 A1 WO 2022154119A1
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- soluble gpc3
- gpc3
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- soluble
- immunoassay
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Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/303—Liver or Pancreas
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4722—Proteoglycans, e.g. aggreccan
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2470/00—Immunochemical assays or immunoassays characterised by the reaction format or reaction type
- G01N2470/04—Sandwich assay format
Definitions
- the present invention relates to a method for treating a soluble GPC3-containing sample in an immunoassay of soluble glypican-3 (GPC3).
- GPC3 is a protein having a molecular weight of about 65 kDa belonging to the glypican family of proteoglycans having a heparan sulfate chain, which is bound to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor present at its C-terminal. .. GPC3 is cleaved between the arginine residue at position 358 and the serine residue at position 359 in the Golgi apparatus by an enzyme called Furin to form an N-terminal fragment (about 40 kDa) and a GPI anchor-containing C-terminal fragment (about 30 kDa). Generate.
- GPI glycosylphosphatidylinositol
- GPC3 is believed to be attached to the cell membrane via a GPI anchor in the form of a full-length protein containing two fragments linked by disulfide bonds.
- Patent Document 1 proposes a method for inspecting a cancer patient by measuring soluble GPC3.
- Patent Document 2 proposes a method for measuring GPC3 using two different antibodies that bind to different epitopes existing in the N-terminal region of GPC3.
- An object of the present invention is to provide a method and a reagent useful for testing soluble GPC3.
- the present inventors have found that in the immunoassay of soluble GPC3, the accuracy of discrimination between a positive sample and a negative sample can be improved by treating the sample with a reducing agent.
- the present inventors also found that in the immunoassay of soluble GPC3, the accuracy of discrimination between a positive sample and a negative sample can be improved and the matrix effect can be reduced by treating the sample with both a reducing agent and a surfactant. , The present invention has been completed.
- the present invention is as follows.
- the method of [3], wherein the surfactant is used at a final concentration of 0.005 to 10% by weight.
- An immunoassay method for soluble GPC3, which comprises the following (a) and (b): (A) Mixing a soluble GPC3-containing sample with a reducing agent; and (b) measuring the amount of soluble GPC3 in the mixture obtained in (a) using one or more antibodies against soluble GPC3. [10] The method of [9], which comprises further mixing the soluble GPC3 containing sample with a surfactant. [11] The method of [9] or [10], wherein the measurement is performed by a sandwich immunoassay using two or more different antibodies against two or more different epitopes in soluble GPC3.
- Soluble GPC3 immunoassay reagent comprising the following (a) and (b): (A) Reducing agent; and (b) One or more antibodies against soluble GPC3.
- the immunoassay reagent of [12] further comprising (c) a surfactant.
- One or more antibodies against soluble GPC3 are two or more different antibodies against two or more different epitopes in soluble GPC3, and the immunoassay reagent is for sandwich immunoassay, [12] or [ 13] Immunoassay reagent.
- the immunoassay reagent according to any one of [12] to [14], wherein the reagent is for diagnosing cancer.
- the present invention in the immunoassay of soluble GPC3, the accuracy of discrimination between a positive sample and a negative sample can be improved. Therefore, the present invention is useful in achieving high diagnostic sensitivity for specific conditions (eg, diseases such as cancer) in the immunoassay of soluble GPC3.
- specific conditions eg, diseases such as cancer
- FIG. 1 is a diagram showing analysis of recognition sites of antibody A and antibody B in an antigen protein by Western blotting (WB).
- WB Western blotting
- antigen proteins cultured supernatants of recombinant human GPC3 (rhGPC3) consisting of amino acid residues 1-559 of human GPC3 and human liver cancer cell HepG2 with high GPC3 expression were used.
- the amounts of protein applied in the lanes were 1.4 ng / lane and 7.2 ng / lane, respectively.
- the present invention provides a method for processing a sample in an immunoassay for soluble GPC3.
- soluble GPC3 refers to soluble GPC3 secreted from GPC3-expressing cells.
- soluble GPC3 derived from any subject can be used.
- Such subjects include, for example, mammals (eg, primates such as humans and monkeys; rodents such as mice, rats and rabbits; hoofed animals such as cows, pigs, goats, horses and sheep, dogs). , Cats and other meats), birds (eg, chickens).
- the subject is a mammal such as a human.
- GPC3 is widely conserved in animals, and its amino acid sequence is highly conserved, especially among mammals. From the point of view of clinical application, the subject is preferably a human. Therefore, soluble GPC3 is preferably human soluble GPC3.
- human soluble GPC3 is produced by cleavage between the arginine residue at position 358 and the serine residue at position 359 in the human GPC3 protein (accession number: P51654.1) consisting of 580 amino acid residues. It is a soluble full-length GPC3 released by cleavage of the N-terminal fragment or the GPI anchor present at the C-terminal of the human GPC3 protein.
- the soluble full-length GPC3 for example, the N-terminal fragment and the C-terminal fragment produced by cleavage between the arginine residue at position 358 and the serine residue at position 359 in the human GPC3 protein are linked to each other via a disulfide bond. GPC3 fragments can be mentioned.
- such human soluble GPC3 is (a) an N-terminal fragment consisting of amino acid residues 1 to 358 in the amino acid sequence of SEQ ID NO: 1 or a variant thereof, and (b) the amino acid sequence of SEQ ID NO: 1.
- the N-terminal fragment consisting of the amino acid residues at positions 1 to 358 or a variant thereof and the soluble C-terminal fragment consisting of the amino acid residues at positions 359 to 560 in the amino acid sequence of SEQ ID NO: 1 or a variant thereof were linked by a disulfide bond.
- Such variants are one or more amino acids that can occur naturally between races and / or individuals with respect to (a) the N-terminal fragment or variant thereof, or (b) soluble full-length GPC3 or variants thereof. Mutations in residues (eg, substitutions, insertions, deletions) have been introduced. The number of mutations in amino acid residues in such mutants is, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 15, even more preferably 1 to 10, and particularly preferably 1. It may be 2, 3, 4, or 5.
- the immunoassay refers to an immunoassay of soluble GPC3 using one or more antibodies against soluble GPC3.
- the antibody include a polyclonal antibody and a monoclonal antibody.
- the antibody is a monoclonal antibody.
- Antibodies can also be identified by isotype. Such isotypes include, for example, IgG, IgM, IgA, IgD, IgE, and IgY.
- the antibody is IgG, IgM, or IgA, more preferably IgG, or IgM, and even more preferably IgG.
- the antibody may further be a chimeric antibody, a humanized antibody, or a human antibody.
- the antibody may also be a full-length antibody or fragment thereof comprising a heavy chain and a light chain containing a variable region and a constant region, respectively.
- Antibody fragments include, for example, F (ab') 2 , Fab', Fab, and Fv. Further, the antibody may be a single chain antibody (scFv) or a VHH antibody.
- the antibody against soluble GPC3 used in the present invention is not particularly limited as long as it is 1 or more, and may be 1, 2, 3, 4, or 5.
- two or more antibodies against soluble GPC3 can recognize the same or different epitopes.
- such two or more antibodies may recognize different epitopes. Since various epitopes available in the immunoassay of soluble GPC3 (eg, two or more different epitopes available in the sandwich assay) and antibodies to the epitope are known (eg, WO 2015 / (See 09928, 2004/038420, 2004/02723)), such known epitopes and antibodies to them may be used in the present invention.
- an antibody against soluble GPC3 is commercially available, a commercially available antibody can also be used in the present invention. From the viewpoint of simple immunoassay and the like, it is preferable to use one or two antibodies against soluble GPC3.
- the antibody against soluble GPC3 used in the present invention is preferably an antibody having an ability to bind to a region in the N-terminal fragment of GPC3, and has an ability to specifically bind to a region in the N-terminal fragment of GPC3. It is more preferably an antibody.
- the immunoassay can be performed by any immunoassay using one or more antibodies against soluble GPC3.
- immunoassays include, for example, chemiluminescent immunoassay (CLIA) [eg, chemiluminescent enzyme immunoassay (CLEIA)], immunoturbidimetric method (TIA), enzyme-linked immunosorbent assay (EIA) (eg, direct ELISA, etc.). Indirect ELISA, competitive ELISA), radioimmunoassay (RIA), latex aggregation reaction, fluorescent immunoassay (FIA), and immunochromatography, western blotting, immunostaining.
- CLIA chemiluminescent immunoassay
- TIA immunoturbidimetric method
- EIA enzyme-linked immunosorbent assay
- Indirect ELISA, competitive ELISA radioimmunoassay
- RIA radioimmunoassay
- FIA fluorescent immunoassay
- immunochromatography western blotting, immuno
- the immunoassay can also be performed by any immunoassay using two or more antibodies against soluble GPC3, including a labeled antibody against soluble GPC3 and a solid phase antibody.
- a labeled antibody is an antibody labeled with a labeling substance or an antibody labeled with a labeling substance in the step of immunoassay.
- Labeling substances include, for example, fluorescent substances, luminescent substances, dyes, and enzymes.
- a solid phase antibody is an antibody that is immobilized on the solid phase or an antibody that is immobilized on the solid phase in the step of immunoassay.
- Solid phases include, for example, particles (eg, microparticles, nanoparticles, microbeads, nanobeads, microspheres, nanospheres), supports (eg, membranes), and substrates (eg, plates).
- the solid phase may be a magnetic solid phase (eg, magnetic particles).
- the immunoassay can also be performed in any manner. Such modes include, for example, the direct method, the indirect method, the competitive method, and the sandwich method.
- the immunoassay may be performed by a sandwich immunoassay using two or more different antibodies against two or more different epitopes of soluble GPC3.
- two or more antibodies including a labeled antibody against soluble GPC3 and a solid phase antibody are used as different two or more antibodies against two or more different epitopes of soluble GPC3.
- the immunoassay may be performed by a sandwich immunoassay using two antibodies (labeled antibody and solid phase antibody) against two different epitopes in soluble GPC3.
- Two antibodies against two different epitopes of soluble GPC3 are two antibodies against different epitopes of the C-terminal fragment, even if they are two antibodies against different epitopes of the N-terminal fragment. May be.
- such two types of antibodies may be a combination of one type of antibody against the epitope of the N-terminal fragment and one type of antibody against the epitope of the C-terminal fragment.
- such two antibodies are two antibodies against different epitopes of the N-terminal fragment.
- the method for treating a soluble GPC3-containing sample according to the present invention includes mixing the soluble GPC3-containing sample with a reducing agent. As a result, a mixed solution of the soluble GPC3-containing sample and the reducing agent is produced.
- the soluble GPC3-containing sample is any sample containing the above-mentioned soluble GPC3.
- the soluble GPC3-containing sample include a liquid sample obtained from the subject (eg, blood, lymph, urine, milk, saliva, tears), a tissue extract sample obtained from the subject, and a washing solution that can be recovered from the subject. Specimens (eg, obtained by washing mucous tissue such as bronchi), specimens obtained from cell cultures derived from subjects, and specimens containing recombinant soluble GPC3 (eg, soluble GPC3 standard), and these. Examples thereof include liquid samples obtained by processing (eg, fractionating) a sample. From the viewpoint of easy acquisition of a sample containing abundant soluble GPC3, the soluble GPC3-containing sample is preferably a blood sample (eg, whole blood, serum, plasma).
- the soluble GPC3 containing sample may be a sample obtained from a subject in a particular state.
- a subject include a subject suffering from a specific disease and a subject who may have a specific disease.
- Specific diseases include, for example, cancer (eg, liver cancer, prostate cancer, malignant melanoma), liver disease (eg, hepatitis, cirrhosis) (eg, WO 2004/038420; WO 2007).
- cancer eg, liver cancer, prostate cancer, malignant melanoma
- liver disease eg, hepatitis, cirrhosis
- WO 2004/038420 eg, WO 2004/038420; WO 2007.
- 081790 International Publication No. 2005/039380
- Detection of glypican-3-specific CTLs in chronic hepatitis and liver cirrhosis Oncology Reports 22, p. 149-54, p. 149-54.
- any reducing agent can be used.
- reducing agents include 2- (dimethylamino) ethanethiol (DEAET), tris (2-carboxyethyl) phosphine (TCEP), 2-mercaptoethylamine, 2-mercaptoethanol, dithiothreitol, and thioglycerol.
- TCEP 2,2-carboxyethyl) phosphine
- 2-mercaptoethylamine 2-mercaptoethanol
- dithiothreitol dithiothreitol
- thioglycerol thioglycerol
- Sodium sulfite, and borohydride and salts thereof.
- the salt include metal salts (eg, monovalent metal salts such as sodium salt and potassium salt, and divalent metal salts such as calcium salt and magnesium salt) and inorganic salts (eg, fluoride, chloride, etc.).
- halide salts such as iodide, and ammonium salts
- organic salts eg, ammonium salts substituted with alkyl groups
- acid addition salts eg, sulfuric acid, hydrochloric acid, hydrobromic acid, nitrate, phosphoric acid.
- Salts with inorganic acids such as acetic acid, oxalic acid, lactic acid, citric acid, trifluoromethanesulfonic acid, salts with organic acids such as trifluoroacetic acid). From the standpoint of using a reducing agent that is highly stable in solution, DEAET, TCEP, or salts thereof are preferred.
- the reducing agent can be used at a final concentration that can reduce the detection signal intensity (eg, count) from negative samples.
- the final concentration is the concentration at the time of mixing. Therefore, the final concentration can also be expressed as the concentration in the mixed solution produced by mixing.
- Such a final concentration is, for example, 0.05 to 1,000 mM, preferably 0.5 to 500 mM, more preferably 1 to 300 mM, and even more preferably 3 to 200 mM.
- Mixing the soluble GPC3 containing sample and the reducing agent may include further mixing the soluble GPC3 containing sample with the surfactant. In such a case, a mixed solution of a soluble GPC3-containing sample, a reducing agent, and a surfactant is produced.
- Examples of the surfactant include nonionic surfactants, amphoteric surfactants, anionic surfactants, and cationic surfactants, and salts thereof.
- the salt is similar to that described above.
- Examples of the nonionic surfactant include polyoxyethylene sorbitan fatty acid ester, polyoxyethylene alkyl phenyl ether, and polyoxyethylene alkyl ether.
- Examples of the polyoxyethylene sorbitan fatty acid ester include polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monopalmitate (Tween 40), polyoxyethylene sorbitan monostearate (Tween 60), and polyoxyethylene sorbitan monoole. Ate (Tween 80) can be mentioned.
- polyoxyethylene alkyl phenyl ether examples include polyoxyethylene (10) octylphenyl ether (Triton X-100), polyoxyethylene (8) octylphenyl ether (Triton X-114), and polyoxyethylene (30) octyl. Examples thereof include phenyl ether (Triton X-305) and polyoxyethylene (40) octyl phenyl ether (Triton X-405).
- polyoxyethylene alkyl ether examples include polyoxyethylene (23) lauryl ether (Brij35) and polyoxyethylene (20) cetyl ether (Brij58).
- nonionic surfactant examples include polyoxyethylene sorbitan monolaurate (Tween 20) and polyoxyethylene (10) octylphenyl ether (Triton X-100).
- examples of the zwitterionic surfactant include a sulfobetaine type surfactant.
- the sulfobetaine-type surfactant examples include 3-[(3-colamidpropyl) dimethylammonio] -1-propanesulfonate (CHAPS) and 3- [3-colamidpropyl] dimethylammonio) -2.
- CHPSO Hydrophilicity-N-(CH 3 )-2-Hydroxypropanesulfonate
- N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate
- N- Hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate can be mentioned.
- Anionic surfactants include, for example, sodium dodecyl sulfate (SDS), sodium N-lauroyl sarcosin (NLS) lithium dodecyl sulfate, sodium dodecylbenzene sulfonate, and deoxycholate.
- Examples of the cationic surfactant include decyltrimethylammonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride, decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, and hexa. Calcyltrimethylammonium bromide can be mentioned.
- the surfactant is a nonionic surfactant, or an amphoteric surfactant, or a salt thereof.
- the surfactant can be used at a final concentration that can enhance the effect of the reducing agent on reducing the detection signal intensity from the negative sample and / or reduce the matrix effect of the sample.
- the final concentration is the concentration at the time of mixing. Therefore, the final concentration can also be expressed as the concentration in the mixed solution produced by mixing.
- Such a final concentration is, for example, 0.005 to 10% by weight, preferably 0.01 to 9% by weight, more preferably 0.02 to 7.5% by weight, and even more preferably 0.05 to 6% by weight. , Particularly preferably 0.05 to 5% by weight.
- the reducing agent and the surfactant can be used in a ratio capable of enhancing the effect in reducing the detection signal intensity from the negative sample and / or reducing the matrix effect of the sample.
- a ratio can be defined by the concentration range of the surfactant per 1 mM of the reducing agent.
- the concentration range of the surfactant per 1 mM of the reducing agent is, for example, 0.000025 to 3% by weight, preferably 0.00005 to 0.1% by weight, more preferably 0.0005 to 0.01% by weight, and even more preferably. Is 0.005 to 0.05% by weight.
- the mixing can be performed simultaneously or separately.
- the soluble GPC3 containing sample can be mixed with a mixture of reducing agent and surfactant. If the mixing is done separately, the soluble GPC3 containing sample may be mixed first with the reducing agent and then with the surfactant, or first with the surfactant and then with the reducing agent.
- the reducing agent and / or surfactant can be used by dissolving it in an aqueous solution.
- an aqueous solution include water (eg, distilled water, sterilized water, sterilized distilled water, pure water), and a buffer solution.
- the buffer solution include phosphate buffer solution, MES buffer solution, citric acid buffer solution, Tris buffer solution, carbon dioxide buffer solution, HPEPS buffer solution, and MOPS buffer solution.
- the pH of the buffer solution varies depending on factors such as the type and concentration of the reducing agent, but from the viewpoint of improving the effect of the reducing agent, it is 1.0 to 9.0 (preferably 4.0 to 6.0). ) May be.
- the aqueous solution is preferably a buffer solution from the viewpoint of stably exerting the effect of the reducing agent in a desired pH range.
- the aqueous solution may contain other components such as an organic solvent (eg, alcohol).
- the ratio of the volume to be mixed with the soluble GPC3-containing sample and the aqueous solution containing the reducing agent and / or the surfactant is, for example, 10: 1. It is ⁇ 1:10, preferably 5: 1 to 1: 5, and more preferably 2: 1 to 1: 2.
- Mixing is carried out under conditions sufficient for processing the sample with the reducing agent alone or with both the reducing agent and the surfactant.
- Such temperature conditions are, for example, 15 to 60 ° C., preferably 20 to 50 ° C., more preferably 25 to 45 ° C.
- the mixing time is, for example, 30 seconds or less. From the viewpoint of rapid processing and the like, the mixing time is preferably 20 seconds or less, more preferably 15 seconds or less.
- the method of the present invention may include further incubating the mixed solution after mixing.
- Incubation time is determined by the type and concentration of reducing agent, the presence or absence of a surfactant in combination, the type and concentration of surfactant, the mixing time, and the desired reduction in detection signal intensity, and measurement using an antibody (immunoassay). When is performed, it varies depending on factors such as the time required for it, but is, for example, 120 minutes or less, preferably 60 minutes or less, and more preferably 30 minutes or less. From the viewpoint of rapid treatment and the like, the incubation time is even more preferably 20 minutes or less, and particularly preferably 10 minutes or less, or 5 minutes or less.
- the incubation temperature is similar to the temperature conditions in the mixing described above.
- the present invention also provides an immunoassay method for soluble GPC3, which comprises the following (a) and (b): (A) Mixing a soluble GPC3-containing sample with a reducing agent; and (b) measuring the amount of soluble GPC3 in the mixture obtained in (a) using one or more antibodies against soluble GPC3.
- Step (a) can be performed in the same manner as the method for treating a soluble GPC3-containing sample according to the present invention.
- Step (b) can be performed by the immunoassay described above.
- Steps (a) and (b) can be performed in parallel or separately.
- a reducing agent can be obtained by simultaneously mixing a soluble GPC3 containing sample, a reducing agent (and a surfactant), and one or more antibodies against the soluble GPC3.
- Treatment of the soluble GPC3-containing sample with (and surfactant) and antigen-antibody reaction can be performed simultaneously.
- the soluble GPC3-containing sample should be sufficiently treated with a reducing agent (and surfactant), and then one of the soluble GPC3-containing samples. It is preferable to measure the amount of soluble GPC3 by an antigen-antibody reaction using the above antibody. Further, when the antibody used in the present invention contains a disulfide bond, if the antibody and the reducing agent coexist for a long time, the reducing agent destroys the antibody by cleaving the disulfide bond in the antibody, which affects the measurement accuracy of the immunoassay. Can be.
- steps (a) and (b) are preferably performed separately.
- steps (a) and (b) are also preferable to carry out steps (a) and (b) in parallel.
- the present invention further provides an immunoassay reagent for soluble GPC3, which comprises the following (a) and (b): (A) Reducing agent; and (b) One or more antibodies against soluble GPC3.
- the reagent of the present invention may further contain (c) a surfactant.
- the reagents of the invention are such that one or more antibodies against soluble GPC3 are two or more different antibodies against two or more different epitopes in soluble GPC3, and the reagents are for sandwich immunoassays. It may be a reagent that is.
- such reagents include labeled and / or solid phase antibodies against soluble GPC3 as the two or more antibodies described above.
- such reagents may include a labeling substance and / or a solid phase if it does not contain a labeling antibody labeled with a labeling substance and / or a solid phase antibody immobilized on a solid phase.
- Labeling substances include, for example, fluorescent substances, luminescent substances, dyes, and enzymes.
- the labeling substance is an enzyme
- a reagent produces a substrate of the enzyme (eg, a substrate that produces a detection signal, or a substrate that is converted by the enzyme into a product that produces a detection signal, or a detection signal. It may also include a substrate, or a substrate in a reaction that can be coupled to another enzymatic reaction that utilizes a substrate that is converted into a product that produces a detection signal by the enzyme.
- the solid phase is similar to that described above.
- the reagent of the present invention can be used for determining a specific state (eg, diagnosing a disease). Specific conditions (eg, disease) are similar to those described above.
- the reagent of the present invention can be used for diagnosing cancers such as liver cancer and prostate cancer.
- a sample buffer for SDS-PAGE was added to the rhGPC3 and HepG2 culture supernatants, and the rhGPC3 and HepG2 culture supernatants were mixed with a 5-20% polyacrylamide gel so as to have 1.4 ng protein / lane and 7.2 ng protein / lane, respectively.
- Applied to (Superset Ace 5-20%, WAKO).
- electrophoresis (30 mA, 60 minutes)
- the protein was transferred to a blotting membrane (Imoviron ISEC, Merck Millipore) using a Transblot® SD semi-dry electrophoresis transfer cell (Bio-Rad) (15 V, 60 minutes). ..
- the blotting membrane was lightly washed with TBS-T and then shaken in a blocking solution (0.5% ECL Bl Plusck (GE Healthcare Life Science), 0.5% BSA, TBS-T) for 1 hour at room temperature. .. After washing twice with TBS-T, the blotting membrane was diluted in 1 ⁇ g / mL with a reaction solution (blocking solution diluted 2-fold with TBS-T) in a solution containing anti-GPC3 antibody A and anti-GPC3 antibody B, respectively. Shake overnight at 4 ° C.
- the blotting membrane is shaken in a solution containing POD-labeled anti-mouse F (ab') 2 (Jackson) diluted 20,000 times with the reaction solution for 1 hour, and then with TBS-T. Washed.
- the color development reaction was carried out using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and developed using a Chemilmi imaging system (FUSION SYSTEM, Biller Lumat). The analysis result of Western blotting is shown in FIG.
- both anti-GPC3 antibody A and anti-GPC3 antibody B reacted with a band of 40 kDa corresponding to the N-terminal fragment of GPC3 (the fragment on the N-terminal side of the 358th amino acid residue) (FIG. 1).
- Reference Example 2 Preparation of anti-GPC3 antibody-immobilized particles
- Anti-GPC3 antibody A is added to magnetic particles in 10 mM MES buffer (pH 5.0), and 0.2 mg / mL anti-GPC3 antibody A and 0.01 g / A suspension containing mL magnetic particles was obtained.
- the suspension was incubated at 5 ° C. for 1 hour with gentle stirring to solidify the anti-GPC3 antibody A into magnetic particles. Then, the magnetic particles were magnetized with a magnet, and the magnetic particles were washed with a washing solution (50 mM Tris buffer, 150 mM NaCl, 2.0% BSA, pH 7.2) to obtain anti-GPC3 antibody A immobilized particles. ..
- anti-GPC3 antibody A-immobilized particles were suspended in particle diluent (50 mM Tris buffer, 1 mM EDTA2Na, 0.1% NaN 3 , 2.0% BSA, pH 7.2).
- ALP-labeled anti-GPC3 antibody B was labeled as a diluent (50 mM MES buffer, 150 mM NaCl, 0.3 mM ZnCl 2 , 1 mM MgCl 2 , 0.1% NaN 3 , 2.0% BSA, pH 6.8). Suspended in.
- Reference Example 4 Measurement of soluble GPC3 20 ⁇ L of the pretreatment solution was dispensed into the reaction vessel, and then 20 ⁇ L of the sample was dispensed into the reaction vessel. After incubating the mixed solution of the pretreatment solution and the sample at 37 ° C. for 6.5 minutes, 50 ⁇ L of anti-GPC3 antibody A-immobilized particles were dispensed into the reaction vessel, and the mixed solution was stirred. The mixed solution was incubated at 37 ° C. for 8 minutes to perform B / F separation and washing. After dispensing 50 ⁇ L of ALP-labeled anti-GPC3 antibody B into the reaction vessel, the mixed solution was stirred. The mixed solution was incubated at 37 ° C.
- Example 1 Pretreatment of a sample with a reducing agent DEAET in the measurement of soluble GPC3
- a serum sample derived from a healthy person (negative sample) and a serum sample derived from a liver cancer patient positive for ⁇ -fetoprotein (AFP) (Trina) (Positive sample) was used as a sample for evaluation of soluble GPC3.
- a phosphate buffer solution (10 mM phosphate buffer solution, 6.25-600 mM DEAET, pH 6.0) containing 6.25 mM to 600 mM 2- (dimethylamino) ethanethiol hydrochloride (DEAET) was used. board.
- a 10 mM phosphate buffer solution pH 6.0
- Negative sample, positive sample and buffer sample (10 mM phosphate buffer) were mixed with the pretreatment solution at a volume ratio of 1: 1 and reacted at 37 ° C. for 6.5 minutes (with pretreatment).
- the negative sample, the positive sample and the buffer solution sample were mixed with the control solution and reacted (without pretreatment).
- Table 1 shows (1) reduction rate (%), (2) positive and negative count difference (%), and (3) matrix difference (%) calculated from the counts by the following formula.
- the rate of decrease (%) is an index for evaluating the effect of pretreatment on individual samples.
- the positive and negative count difference (%) is an index for evaluating the difference between the counts of the positive sample and the negative sample due to the pretreatment.
- the matrix difference (%) is an index for evaluating the difference in count between the buffer solution sample and the negative sample.
- the substance contained in the specimen may affect the immune response (matrix effect).
- the reduced matrix effect is useful for the measurement system of soluble GPC3 because it can achieve a true value output independent of the sample species.
- Example 2 Pretreatment of Specimen with Reducing Agent 2MEA in Measurement of Soluble GPC3 Phosphate Buffer Solution (10 mM Phosphate Buffer Solution, 10 mM Phosphate Buffer Solution,) containing 6.25 mM to 100 mM 2-mercaptoethylamine hydrochloride (2MEA) as a pretreatment solution. 6.25 to 100 mM 2MEA, pH 6.0) was used. As a control solution, a 10 mM phosphate buffer solution (pH 6.0) was used. Negative samples, positive samples and buffer samples were mixed with the pretreatment solution at a volume ratio of 1: 1 and reacted at 37 ° C. for 6.5 minutes (with pretreatment). Similarly, the negative sample, the positive sample and the buffer solution sample were mixed with the control solution and reacted (without pretreatment).
- Soluble GPC3 Phosphate Buffer Solution 10 mM Phosphate Buffer Solution, 10 mM Phosphate
- soluble GPC3 was measured by the measuring method described in Reference Example 4. The count of each sample is shown in Table 2. Further, in the same manner as in Example 1, the reduction rate (%), the positive and negative count difference (%), and the matrix difference (%) were calculated.
- Example 3 Pretreatment of Samples with Reducing Agent TCEP in Measurement of Soluble GPC3 Phosphate Buffer (10 mM phosphorus) containing 6.25 mM-100 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) as a pretreatment solution.
- Acid buffer, 6.25-100 mM TCEP, pH 6.0 was used.
- As a control solution a 10 mM phosphate buffer solution (pH 6.0) was used.
- Negative samples, positive samples and buffer samples were mixed with the pretreatment solution at a volume ratio of 1: 1 and reacted at 37 ° C. for 6.5 minutes (with pretreatment). Similarly, the negative sample, the positive sample and the buffer solution sample were mixed with the control solution and reacted (without pretreatment).
- soluble GPC3 was measured by the measuring method described in Reference Example 4. The count of each sample is shown in Table 3. Further, in the same manner as in Example 1, the reduction rate (%), the positive and negative count difference (%), and the matrix difference (%) were calculated.
- Example 4 Pretreatment of a sample with a combination of a reducing agent and a surfactant in the measurement of soluble GPC3
- the phosphate buffer solution (pH 6.0) of Test Examples 2 to 11 shown in Table 4A was used.
- the phosphate buffers of Test Examples 2 to 11 are DEAET as a reducing agent and 3-[(3-colamidepropyl) dimethylammonio] -1-propanesulfonate (CHAPS) as a surfactant, or polyoxyethylene sorbitan. It contained monolaurate (Tween 20).
- the 10 mM phosphate buffer solution (pH 6.0) of Test Example 1 shown in Table 4A was used.
- Negative samples, positive samples and buffer samples were mixed with the pretreatment solution at a volume ratio of 1: 1 and reacted at 37 ° C. for 6.5 minutes (with pretreatment). Similarly, the negative sample, the positive sample and the buffer solution sample were mixed with the control solution and reacted (without pretreatment).
- soluble GPC3 was measured by the measuring method described in Reference Example 4. The count of each sample is shown in Table 4B. Further, in the same manner as in Example 1, the reduction rate (%), the positive and negative count difference (%), and the matrix difference (%) were calculated.
- the matrix difference became closer to 100%, and the matrix effect was further reduced.
- the pretreatment of the sample with the combination of the reducing agent and the surfactant can improve the accuracy of discriminating between the positive sample and the negative sample in the measurement of soluble GPC3, and can reduce the matrix effect of the sample. Therefore, it was confirmed that the pretreatment of the sample with the combination of the reducing agent and the surfactant is useful for the measurement of soluble GPC3.
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Abstract
The present invention provides a method and reagent useful for testing for soluble GPC3. More specifically, the present invention provides (1), (2), and (3). (1) A processing method for specimens in soluble GPC3 immunoassays, that includes mixing a soluble GPC3-containing specimen with a reducing agent. (2) A soluble GPC3 immunoassay method that includes (a) and (b). (a) Mixing the soluble GPC3-containing specimen with a reducing agent; and (b) using at least one type of antibody against soluble GPC3 and measuring the amount of soluble GPC3 in the mixed liquid obtained in (a). (3) A soluble GPC3 immunoassay method that includes (a) and (b). (a) A reducing agent; and (b) at least one antibody against soluble GPC3.
Description
本発明は、可溶性グリピカン－３（ＧＰＣ３）のイムノアッセイにおける可溶性ＧＰＣ３含有検体の処理方法などに関する。
The present invention relates to a method for treating a soluble GPC3-containing sample in an immunoassay of soluble glypican-3 (GPC3).
ＧＰＣ３は、そのＣ末端に存在するグリコシルフォスファチジルイノシトール（ＧＰＩ）アンカーを介して細胞膜に結合している、ヘパラン硫酸鎖を有するプロテオグリカンであるグリピカンファミリーに属する約６５ｋＤａの分子量を有するタンパク質である。ＧＰＣ３は、ゴルジ体においてＦｕｒｉｎと呼ばれる酵素により３５８位のアルギニン残基と３５９位のセリン残基との間で切断されてＮ末端断片（約４０ｋＤａ）およびＧＰＩアンカー含有Ｃ末端断片（約３０ｋＤａ）を生成する。しかし、このような２つの断片は通常、ジスルフィド結合により連結していることが確認されている。したがって、ＧＰＣ３は、ジスルフィド結合により連結された２つの断片を含む全長型タンパク質の形態において、ＧＰＩアンカーを介して細胞膜に結合していると考えられている。
GPC3 is a protein having a molecular weight of about 65 kDa belonging to the glypican family of proteoglycans having a heparan sulfate chain, which is bound to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor present at its C-terminal. .. GPC3 is cleaved between the arginine residue at position 358 and the serine residue at position 359 in the Golgi apparatus by an enzyme called Furin to form an N-terminal fragment (about 40 kDa) and a GPI anchor-containing C-terminal fragment (about 30 kDa). Generate. However, it has been confirmed that such two fragments are usually linked by a disulfide bond. Therefore, GPC3 is believed to be attached to the cell membrane via a GPI anchor in the form of a full-length protein containing two fragments linked by disulfide bonds.
ＧＰＣ３はまた、肝臓癌等の癌において特異的な発現が認められるタンパク質であるため、ＧＰＣ３を標的とする癌患者の検査に有用な方法の開発が模索されている。例えば、特許文献１では、可溶性ＧＰＣ３の測定による癌患者の検査方法が提案されている。また、特許文献２では、ＧＰＣ３のＮ末端領域に存在する異なるエピトープに結合する異なる２つの抗体を用いるＧＰＣ３の測定方法が提案されている。
Since GPC3 is also a protein whose specific expression is observed in cancers such as liver cancer, the development of a method useful for testing cancer patients targeting GPC3 is being sought. For example, Patent Document 1 proposes a method for inspecting a cancer patient by measuring soluble GPC3. Further, Patent Document 2 proposes a method for measuring GPC3 using two different antibodies that bind to different epitopes existing in the N-terminal region of GPC3.
本発明の課題は、可溶性ＧＰＣ３の検査に有用な方法および試薬を提供することである。
An object of the present invention is to provide a method and a reagent useful for testing soluble GPC3.
本発明者らは、鋭意検討した結果、可溶性ＧＰＣ３のイムノアッセイにおいて、検体を還元剤で処理することにより、陽性検体と陰性検体との判別精度を向上できることを見出した。本発明者らはまた、可溶性ＧＰＣ３のイムノアッセイにおいて、検体を還元剤および界面活性剤の双方で処理することにより、陽性検体と陰性検体との判別精度を向上できること、およびマトリックス効果を低減できることを見出し、本発明を完成するに至った。
As a result of diligent studies, the present inventors have found that in the immunoassay of soluble GPC3, the accuracy of discrimination between a positive sample and a negative sample can be improved by treating the sample with a reducing agent. The present inventors also found that in the immunoassay of soluble GPC3, the accuracy of discrimination between a positive sample and a negative sample can be improved and the matrix effect can be reduced by treating the sample with both a reducing agent and a surfactant. , The present invention has been completed.
すなわち、本発明は、以下のとおりである。
〔１〕可溶性ＧＰＣ３含有検体を還元剤と混合することを含む、可溶性ＧＰＣ３のイムノアッセイにおける可溶性ＧＰＣ３含有検体の処理方法。
〔２〕還元剤が、０．０５～１，０００ｍＭの終濃度で用いられる、〔１〕の方法。
〔３〕可溶性ＧＰＣ３含有検体を界面活性剤とさらに混合することを含む、〔１〕または〔２〕の方法。
〔４〕界面活性剤が、０．００５～１０重量％の終濃度で用いられる、〔３〕の方法。
〔５〕イムノアッセイが、可溶性ＧＰＣ３中の異なる２種以上のエピトープに対する異なる２種以上の抗体を用いるサンドイッチイムノアッセイである、〔１〕～〔４〕のいずれかの方法。
〔６〕可溶性ＧＰＣ３含有検体が血液検体である、〔１〕～〔５〕のいずれかの方法。
〔７〕可溶性ＧＰＣ３含有検体が、癌に罹患している被験体から得られる、〔１〕～〔６〕のいずれかの方法。
〔８〕癌が肝臓癌である、〔７〕の方法。
〔９〕下記（ａ）および（ｂ）を含む、可溶性ＧＰＣ３のイムノアッセイ方法：
（ａ）可溶性ＧＰＣ３含有検体を還元剤と混合すること；および
（ｂ）（ａ）で得られた混合液中の可溶性ＧＰＣ３量を、可溶性ＧＰＣ３に対する１種以上の抗体を用いて測定すること。
〔１０〕可溶性ＧＰＣ３含有検体を界面活性剤とさらに混合することを含む、〔９〕の方法。
〔１１〕測定が、可溶性ＧＰＣ３中の異なる２種以上のエピトープに対する異なる２種以上の抗体を用いるサンドイッチイムノアッセイにより行われる、〔９〕または〔１０〕の方法。
〔１２〕下記（ａ）および（ｂ）を含む、可溶性ＧＰＣ３のイムノアッセイ試薬：
（ａ）還元剤；および
（ｂ）可溶性ＧＰＣ３に対する１種以上の抗体。
〔１３〕さらに（ｃ）界面活性剤を含む、〔１２〕のイムノアッセイ試薬。
〔１４〕可溶性ＧＰＣ３に対する１種以上の抗体が、可溶性ＧＰＣ３中の異なる２種以上のエピトープに対する異なる２種以上の抗体であり、かつ、前記イムノアッセイ試薬がサンドイッチイムノアッセイ用である、〔１２〕または〔１３〕のイムノアッセイ試薬。
〔１５〕前記試薬が癌の診断用である、〔１２〕～〔１４〕のいずれかのイムノアッセイ試薬。 That is, the present invention is as follows.
[1] A method for treating a soluble GPC3-containing sample in an immunoassay of soluble GPC3, which comprises mixing the soluble GPC3-containing sample with a reducing agent.
[2] The method of [1], wherein the reducing agent is used at a final concentration of 0.05 to 1,000 mM.
[3] The method of [1] or [2], which comprises further mixing a soluble GPC3 containing sample with a surfactant.
[4] The method of [3], wherein the surfactant is used at a final concentration of 0.005 to 10% by weight.
[5] The method according to any one of [1] to [4], wherein the immunoassay is a sandwich immunoassay using two or more different antibodies against two or more different epitopes in soluble GPC3.
[6] The method according to any one of [1] to [5], wherein the soluble GPC3-containing sample is a blood sample.
[7] The method according to any one of [1] to [6], wherein the soluble GPC3-containing sample is obtained from a subject suffering from cancer.
[8] The method of [7], wherein the cancer is liver cancer.
[9] An immunoassay method for soluble GPC3, which comprises the following (a) and (b):
(A) Mixing a soluble GPC3-containing sample with a reducing agent; and (b) measuring the amount of soluble GPC3 in the mixture obtained in (a) using one or more antibodies against soluble GPC3.
[10] The method of [9], which comprises further mixing the soluble GPC3 containing sample with a surfactant.
[11] The method of [9] or [10], wherein the measurement is performed by a sandwich immunoassay using two or more different antibodies against two or more different epitopes in soluble GPC3.
[12] Soluble GPC3 immunoassay reagent comprising the following (a) and (b):
(A) Reducing agent; and (b) One or more antibodies against soluble GPC3.
[13] The immunoassay reagent of [12] further comprising (c) a surfactant.
[14] One or more antibodies against soluble GPC3 are two or more different antibodies against two or more different epitopes in soluble GPC3, and the immunoassay reagent is for sandwich immunoassay, [12] or [ 13] Immunoassay reagent.
[15] The immunoassay reagent according to any one of [12] to [14], wherein the reagent is for diagnosing cancer.
〔１〕可溶性ＧＰＣ３含有検体を還元剤と混合することを含む、可溶性ＧＰＣ３のイムノアッセイにおける可溶性ＧＰＣ３含有検体の処理方法。
〔２〕還元剤が、０．０５～１，０００ｍＭの終濃度で用いられる、〔１〕の方法。
〔３〕可溶性ＧＰＣ３含有検体を界面活性剤とさらに混合することを含む、〔１〕または〔２〕の方法。
〔４〕界面活性剤が、０．００５～１０重量％の終濃度で用いられる、〔３〕の方法。
〔５〕イムノアッセイが、可溶性ＧＰＣ３中の異なる２種以上のエピトープに対する異なる２種以上の抗体を用いるサンドイッチイムノアッセイである、〔１〕～〔４〕のいずれかの方法。
〔６〕可溶性ＧＰＣ３含有検体が血液検体である、〔１〕～〔５〕のいずれかの方法。
〔７〕可溶性ＧＰＣ３含有検体が、癌に罹患している被験体から得られる、〔１〕～〔６〕のいずれかの方法。
〔８〕癌が肝臓癌である、〔７〕の方法。
〔９〕下記（ａ）および（ｂ）を含む、可溶性ＧＰＣ３のイムノアッセイ方法：
（ａ）可溶性ＧＰＣ３含有検体を還元剤と混合すること；および
（ｂ）（ａ）で得られた混合液中の可溶性ＧＰＣ３量を、可溶性ＧＰＣ３に対する１種以上の抗体を用いて測定すること。
〔１０〕可溶性ＧＰＣ３含有検体を界面活性剤とさらに混合することを含む、〔９〕の方法。
〔１１〕測定が、可溶性ＧＰＣ３中の異なる２種以上のエピトープに対する異なる２種以上の抗体を用いるサンドイッチイムノアッセイにより行われる、〔９〕または〔１０〕の方法。
〔１２〕下記（ａ）および（ｂ）を含む、可溶性ＧＰＣ３のイムノアッセイ試薬：
（ａ）還元剤；および
（ｂ）可溶性ＧＰＣ３に対する１種以上の抗体。
〔１３〕さらに（ｃ）界面活性剤を含む、〔１２〕のイムノアッセイ試薬。
〔１４〕可溶性ＧＰＣ３に対する１種以上の抗体が、可溶性ＧＰＣ３中の異なる２種以上のエピトープに対する異なる２種以上の抗体であり、かつ、前記イムノアッセイ試薬がサンドイッチイムノアッセイ用である、〔１２〕または〔１３〕のイムノアッセイ試薬。
〔１５〕前記試薬が癌の診断用である、〔１２〕～〔１４〕のいずれかのイムノアッセイ試薬。 That is, the present invention is as follows.
[1] A method for treating a soluble GPC3-containing sample in an immunoassay of soluble GPC3, which comprises mixing the soluble GPC3-containing sample with a reducing agent.
[2] The method of [1], wherein the reducing agent is used at a final concentration of 0.05 to 1,000 mM.
[3] The method of [1] or [2], which comprises further mixing a soluble GPC3 containing sample with a surfactant.
[4] The method of [3], wherein the surfactant is used at a final concentration of 0.005 to 10% by weight.
[5] The method according to any one of [1] to [4], wherein the immunoassay is a sandwich immunoassay using two or more different antibodies against two or more different epitopes in soluble GPC3.
[6] The method according to any one of [1] to [5], wherein the soluble GPC3-containing sample is a blood sample.
[7] The method according to any one of [1] to [6], wherein the soluble GPC3-containing sample is obtained from a subject suffering from cancer.
[8] The method of [7], wherein the cancer is liver cancer.
[9] An immunoassay method for soluble GPC3, which comprises the following (a) and (b):
(A) Mixing a soluble GPC3-containing sample with a reducing agent; and (b) measuring the amount of soluble GPC3 in the mixture obtained in (a) using one or more antibodies against soluble GPC3.
[10] The method of [9], which comprises further mixing the soluble GPC3 containing sample with a surfactant.
[11] The method of [9] or [10], wherein the measurement is performed by a sandwich immunoassay using two or more different antibodies against two or more different epitopes in soluble GPC3.
[12] Soluble GPC3 immunoassay reagent comprising the following (a) and (b):
(A) Reducing agent; and (b) One or more antibodies against soluble GPC3.
[13] The immunoassay reagent of [12] further comprising (c) a surfactant.
[14] One or more antibodies against soluble GPC3 are two or more different antibodies against two or more different epitopes in soluble GPC3, and the immunoassay reagent is for sandwich immunoassay, [12] or [ 13] Immunoassay reagent.
[15] The immunoassay reagent according to any one of [12] to [14], wherein the reagent is for diagnosing cancer.
本発明によれば、可溶性ＧＰＣ３のイムノアッセイにおいて、陽性検体と陰性検体との判別精度を向上できる。したがって、本発明は、可溶性ＧＰＣ３のイムノアッセイにおいて、特定の状態（例、癌等の疾患）に対する高い診断感度の実現に有用である。
According to the present invention, in the immunoassay of soluble GPC3, the accuracy of discrimination between a positive sample and a negative sample can be improved. Therefore, the present invention is useful in achieving high diagnostic sensitivity for specific conditions (eg, diseases such as cancer) in the immunoassay of soluble GPC3.
本発明は、可溶性ＧＰＣ３のイムノアッセイにおける検体の処理方法を提供する。
The present invention provides a method for processing a sample in an immunoassay for soluble GPC3.
本発明では、可溶性ＧＰＣ３は、ＧＰＣ３発現細胞から分泌される可溶性ＧＰＣ３をいう。可溶性ＧＰＣ３としては、任意の被験体由来の可溶性ＧＰＣ３を利用することができる。このような被験体としては、例えば、哺乳動物（例、ヒト、サル等の霊長類；マウス、ラット、ウサギ等の齧歯類；ウシ、ブタ、ヤギ、ウマ、ヒツジ等の有蹄類、イヌ、ネコ等の食肉類）、鳥類（例、ニワトリ）が挙げられる。好ましくは、被験体は、ヒト等の哺乳動物である。ＧＰＣ３は、動物において広く保存されており、特に哺乳動物間ではそのアミノ酸配列が高度に保存されている。臨床応用の観点からは、被験体は、好ましくはヒトである。したがって、可溶性ＧＰＣ３は、好ましくはヒト可溶性ＧＰＣ３である。
In the present invention, soluble GPC3 refers to soluble GPC3 secreted from GPC3-expressing cells. As the soluble GPC3, soluble GPC3 derived from any subject can be used. Such subjects include, for example, mammals (eg, primates such as humans and monkeys; rodents such as mice, rats and rabbits; hoofed animals such as cows, pigs, goats, horses and sheep, dogs). , Cats and other meats), birds (eg, chickens). Preferably, the subject is a mammal such as a human. GPC3 is widely conserved in animals, and its amino acid sequence is highly conserved, especially among mammals. From the point of view of clinical application, the subject is preferably a human. Therefore, soluble GPC3 is preferably human soluble GPC3.
好ましくは、ヒト可溶性ＧＰＣ３は、５８０個のアミノ酸残基からなるヒトＧＰＣ３タンパク質（アクセッション番号：Ｐ５１６５４．１）における３５８位のアルギニン残基と３５９位のセリン残基との間の切断により生成するＮ末端断片、またはヒトＧＰＣ３タンパク質のＣ末端に存在するＧＰＩアンカーの切断により遊離する可溶性全長型ＧＰＣ３である。可溶性全長型ＧＰＣ３としては、例えば、ヒトＧＰＣ３タンパク質における３５８位のアルギニン残基と３５９位のセリン残基との間の切断により生成するＮ末端断片とＣ末端断片がジスルフィド結合を介して互いに連結したＧＰＣ３断片が挙げられる。より具体的には、このようなヒト可溶性ＧＰＣ３は、（ａ）配列番号１のアミノ酸配列における１～３５８位のアミノ酸残基からなるＮ末端断片またはそのバリアント、（ｂ）配列番号１のアミノ酸配列における１～３５８位のアミノ酸残基からなるＮ末端断片またはそのバリアントと、配列番号１のアミノ酸配列における３５９～５６０位のアミノ酸残基からなる可溶性Ｃ末端断片またはそのバリアントとがジスルフィド結合によって連結した可溶性全長型ＧＰＣ３、あるいは（ｃ）人種間および／または個人間において天然に生じ得るそれらの変異体である。このような変異体は、（ａ）Ｎ末端断片またはそのバリアント、または（ｂ）可溶性全長型ＧＰＣ３またはそのバリアントに対して、人種間および／または個人間において天然に生じ得る１個以上のアミノ酸残基の変異（例、置換、挿入、欠失）が導入されたものである。このような変異体におけるアミノ酸残基の変異の個数は、例えば１～３０個、好ましくは１～２０個、より好ましくは１～１５個、さらにより好ましくは１～１０個、特に好ましくは１個、２個、３個、４個又は５個であってもよい。
Preferably, human soluble GPC3 is produced by cleavage between the arginine residue at position 358 and the serine residue at position 359 in the human GPC3 protein (accession number: P51654.1) consisting of 580 amino acid residues. It is a soluble full-length GPC3 released by cleavage of the N-terminal fragment or the GPI anchor present at the C-terminal of the human GPC3 protein. As the soluble full-length GPC3, for example, the N-terminal fragment and the C-terminal fragment produced by cleavage between the arginine residue at position 358 and the serine residue at position 359 in the human GPC3 protein are linked to each other via a disulfide bond. GPC3 fragments can be mentioned. More specifically, such human soluble GPC3 is (a) an N-terminal fragment consisting of amino acid residues 1 to 358 in the amino acid sequence of SEQ ID NO: 1 or a variant thereof, and (b) the amino acid sequence of SEQ ID NO: 1. The N-terminal fragment consisting of the amino acid residues at positions 1 to 358 or a variant thereof and the soluble C-terminal fragment consisting of the amino acid residues at positions 359 to 560 in the amino acid sequence of SEQ ID NO: 1 or a variant thereof were linked by a disulfide bond. Soluble full-length GPC3, or (c) variants thereof that can occur naturally between races and / or individuals. Such variants are one or more amino acids that can occur naturally between races and / or individuals with respect to (a) the N-terminal fragment or variant thereof, or (b) soluble full-length GPC3 or variants thereof. Mutations in residues (eg, substitutions, insertions, deletions) have been introduced. The number of mutations in amino acid residues in such mutants is, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 15, even more preferably 1 to 10, and particularly preferably 1. It may be 2, 3, 4, or 5.
本発明では、イムノアッセイとは、可溶性ＧＰＣ３に対する１種以上の抗体を用いる可溶性ＧＰＣ３のイムノアッセイをいう。抗体としては、例えば、ポリクローナル抗体、およびモノクローナル抗体が挙げられる。好ましくは、抗体は、モノクローナル抗体である。抗体はまた、アイソタイプにより特定することができる。このようなアイソタイプとしては、例えば、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤ、ＩｇＥ、およびＩｇＹが挙げられる。好ましくは、抗体は、ＩｇＧ、ＩｇＭ、またはＩｇＡであり、より好ましくはＩｇＧ、またはＩｇＭであり、さらにより好ましくはＩｇＧである。抗体はさらに、キメラ抗体、ヒト化抗体、またはヒト抗体であってもよい。抗体はまた、可変領域および定常領域を各々含む重鎖および軽鎖を含む全長抗体もしくはその断片であってもよい。抗体断片としては、例えば、Ｆ（ａｂ’）２、Ｆａｂ’、Ｆａｂ、およびＦｖが挙げられる。また、抗体は、単鎖抗体（ｓｃＦｖ）やＶＨＨ抗体であってもよい。
In the present invention, the immunoassay refers to an immunoassay of soluble GPC3 using one or more antibodies against soluble GPC3. Examples of the antibody include a polyclonal antibody and a monoclonal antibody. Preferably, the antibody is a monoclonal antibody. Antibodies can also be identified by isotype. Such isotypes include, for example, IgG, IgM, IgA, IgD, IgE, and IgY. Preferably, the antibody is IgG, IgM, or IgA, more preferably IgG, or IgM, and even more preferably IgG. The antibody may further be a chimeric antibody, a humanized antibody, or a human antibody. The antibody may also be a full-length antibody or fragment thereof comprising a heavy chain and a light chain containing a variable region and a constant region, respectively. Antibody fragments include, for example, F (ab') 2 , Fab', Fab, and Fv. Further, the antibody may be a single chain antibody (scFv) or a VHH antibody.
本発明で用いられる、可溶性ＧＰＣ３に対する抗体は、１種以上である限り特に限定されず、１種、２種、３種、４種、または５種であってもよい。本発明において可溶性ＧＰＣ３に対する２種以上の抗体が用いられる場合、このような２種以上の抗体は、同一または異なるエピトープを認識することができる。好ましくは、このような２種以上の抗体は、異なるエピトープを認識してもよい。可溶性ＧＰＣ３のイムノアッセイにおいて利用可能なエピトープ（例、サンドイッチアッセイで利用可能な異なる２種以上のエピトープ）、および当該エピトープに対する抗体としては種々のものが知られているので（例、国際公開第２０１５／０９７９２８号、国際公開第２００４／０３８４２０号、国際公開第２００４／０２２７３９号を参照）、本発明では、このような既知のエピトープおよびそれに対する抗体を用いてもよい。また、可溶性ＧＰＣ３に対する抗体は市販されているため、本発明では市販抗体を用いることもできる。簡便なイムノアッセイ等の観点から、可溶性ＧＰＣ３に対する１種または２種の抗体の使用が好ましい。本発明で用いられる、可溶性ＧＰＣ３に対する抗体は、ＧＰＣ３のＮ末端断片中の領域に結合する能力を有する抗体であることが好ましく、ＧＰＣ３のＮ末端断片中の領域に特異的に結合する能力を有する抗体であることがより好ましい。
The antibody against soluble GPC3 used in the present invention is not particularly limited as long as it is 1 or more, and may be 1, 2, 3, 4, or 5. When two or more antibodies against soluble GPC3 are used in the present invention, such two or more antibodies can recognize the same or different epitopes. Preferably, such two or more antibodies may recognize different epitopes. Since various epitopes available in the immunoassay of soluble GPC3 (eg, two or more different epitopes available in the sandwich assay) and antibodies to the epitope are known (eg, WO 2015 / (See 09928, 2004/038420, 2004/02723)), such known epitopes and antibodies to them may be used in the present invention. Moreover, since an antibody against soluble GPC3 is commercially available, a commercially available antibody can also be used in the present invention. From the viewpoint of simple immunoassay and the like, it is preferable to use one or two antibodies against soluble GPC3. The antibody against soluble GPC3 used in the present invention is preferably an antibody having an ability to bind to a region in the N-terminal fragment of GPC3, and has an ability to specifically bind to a region in the N-terminal fragment of GPC3. It is more preferably an antibody.
イムノアッセイは、可溶性ＧＰＣ３に対する１種以上の抗体を用いる任意のイムノアッセイにより行うことができる。このようなイムノアッセイとしては、例えば、化学発光イムノアッセイ（ＣＬＩＡ）〔例、化学発光酵素免疫測定法（ＣＬＥＩＡ）〕、免疫比濁法（ＴＩＡ）、酵素免疫測定法（ＥＩＡ）（例、直接ＥＬＩＳＡ、間接ＥＬＩＳＡ、競合ＥＬＩＳＡ、およびサンドイッチＥＬＩＳＡ）、放射イムノアッセイ（ＲＩＡ）、ラテックス凝集反応法、蛍光イムノアッセイ（ＦＩＡ）、およびイムノクロマトグラフィー法、ウェスタンブロッティング、免疫染色が挙げられる。
The immunoassay can be performed by any immunoassay using one or more antibodies against soluble GPC3. Such immunoassays include, for example, chemiluminescent immunoassay (CLIA) [eg, chemiluminescent enzyme immunoassay (CLEIA)], immunoturbidimetric method (TIA), enzyme-linked immunosorbent assay (EIA) (eg, direct ELISA, etc.). Indirect ELISA, competitive ELISA), radioimmunoassay (RIA), latex aggregation reaction, fluorescent immunoassay (FIA), and immunochromatography, western blotting, immunostaining.
イムノアッセイはまた、可溶性ＧＰＣ３に対する標識抗体および固相抗体を含む、可溶性ＧＰＣ３に対する２種以上の抗体を用いる任意のイムノアッセイにより行うことができる。標識抗体は、標識物質で標識された抗体またはイムノアッセイの工程において標識物質で標識される抗体である。標識物質としては、例えば、蛍光物質、発光物質、色素、および酵素が挙げられる。固相抗体は、固相に固定された抗体またはイムノアッセイの工程で固相に固定される抗体である。固相としては、例えば、粒子（例、マイクロ粒子、ナノ粒子、マイクロビーズ、ナノビーズ、マイクロスフェア、ナノスフェア）、支持体（例、メンブレン）、および基板（例、プレート）が挙げられる。固相は、磁性を有する固相（例、磁性粒子）であってもよい。
The immunoassay can also be performed by any immunoassay using two or more antibodies against soluble GPC3, including a labeled antibody against soluble GPC3 and a solid phase antibody. A labeled antibody is an antibody labeled with a labeling substance or an antibody labeled with a labeling substance in the step of immunoassay. Labeling substances include, for example, fluorescent substances, luminescent substances, dyes, and enzymes. A solid phase antibody is an antibody that is immobilized on the solid phase or an antibody that is immobilized on the solid phase in the step of immunoassay. Solid phases include, for example, particles (eg, microparticles, nanoparticles, microbeads, nanobeads, microspheres, nanospheres), supports (eg, membranes), and substrates (eg, plates). The solid phase may be a magnetic solid phase (eg, magnetic particles).
イムノアッセイはまた、任意の様式において行うことができる。このような様式としては、例えば、直接法、間接法、競合法、およびサンドイッチ法が挙げられる。好ましくは、イムノアッセイは、可溶性ＧＰＣ３の異なる２種以上のエピトープに対する異なる２種以上の抗体を用いるサンドイッチイムノアッセイにより行われてもよい。このようなサンドイッチイムノアッセイでは、可溶性ＧＰＣ３の異なる２種以上のエピトープに対する異なる２種以上の抗体として、可溶性ＧＰＣ３に対する標識抗体および固相抗体を含む２種以上の抗体が用いられる。簡便なイムノアッセイ等の観点から、イムノアッセイは、可溶性ＧＰＣ３中の異なる２種のエピトープに対する２種の抗体（標識抗体および固相抗体）を用いるサンドイッチイムノアッセイにより行われてもよい。可溶性ＧＰＣ３の異なる２種のエピトープに対する２種の抗体（標識抗体および固相抗体）は、Ｎ末端断片の異なるエピトープに対する２種の抗体であっても、Ｃ末端断片の異なるエピトープに対する２種の抗体であってもよい。あるいは、このような２種の抗体は、Ｎ末端断片のエピトープに対する１種の抗体と、Ｃ末端断片のエピトープに対する１種の抗体との組合せであってもよい。好ましくは、このような２種の抗体は、Ｎ末端断片の異なるエピトープに対する２種の抗体である。
The immunoassay can also be performed in any manner. Such modes include, for example, the direct method, the indirect method, the competitive method, and the sandwich method. Preferably, the immunoassay may be performed by a sandwich immunoassay using two or more different antibodies against two or more different epitopes of soluble GPC3. In such a sandwich immunoassay, two or more antibodies including a labeled antibody against soluble GPC3 and a solid phase antibody are used as different two or more antibodies against two or more different epitopes of soluble GPC3. From the viewpoint of a simple immunoassay or the like, the immunoassay may be performed by a sandwich immunoassay using two antibodies (labeled antibody and solid phase antibody) against two different epitopes in soluble GPC3. Two antibodies against two different epitopes of soluble GPC3 (labeled antibody and solid phase antibody) are two antibodies against different epitopes of the C-terminal fragment, even if they are two antibodies against different epitopes of the N-terminal fragment. May be. Alternatively, such two types of antibodies may be a combination of one type of antibody against the epitope of the N-terminal fragment and one type of antibody against the epitope of the C-terminal fragment. Preferably, such two antibodies are two antibodies against different epitopes of the N-terminal fragment.
本発明による可溶性ＧＰＣ３含有検体の処理方法は、可溶性ＧＰＣ３含有検体を還元剤と混合することを含む。これにより、可溶性ＧＰＣ３含有検体および還元剤の混合液が生成する。
The method for treating a soluble GPC3-containing sample according to the present invention includes mixing the soluble GPC3-containing sample with a reducing agent. As a result, a mixed solution of the soluble GPC3-containing sample and the reducing agent is produced.
可溶性ＧＰＣ３含有検体は、上述の可溶性ＧＰＣ３を含有する任意の検体である。可溶性ＧＰＣ３含有検体としては、例えば、被験体から得られる液体検体（例、血液、リンパ液、尿、乳汁、唾液、涙液）、被験体から得られる組織の抽出液検体、被験体から回収できる洗浄液検体（例、気管支等の粘膜組織を洗浄して得られるもの）、被験体由来の細胞培養物から得られる検体、および組換え可溶性ＧＰＣ３を含む検体（例、可溶性ＧＰＣ３標品）、ならびにこれらの検体を処理（例、分画）して得られる液体検体が挙げられる。可溶性ＧＰＣ３を豊富に含む検体の簡便な入手等の観点から、可溶性ＧＰＣ３含有検体は、好ましくは、血液検体（例、全血、血清、血漿）である。
The soluble GPC3-containing sample is any sample containing the above-mentioned soluble GPC3. Examples of the soluble GPC3-containing sample include a liquid sample obtained from the subject (eg, blood, lymph, urine, milk, saliva, tears), a tissue extract sample obtained from the subject, and a washing solution that can be recovered from the subject. Specimens (eg, obtained by washing mucous tissue such as bronchi), specimens obtained from cell cultures derived from subjects, and specimens containing recombinant soluble GPC3 (eg, soluble GPC3 standard), and these. Examples thereof include liquid samples obtained by processing (eg, fractionating) a sample. From the viewpoint of easy acquisition of a sample containing abundant soluble GPC3, the soluble GPC3-containing sample is preferably a blood sample (eg, whole blood, serum, plasma).
特定の実施形態では、可溶性ＧＰＣ３含有検体は、特定の状態にある被験体から得られる検体であってもよい。このような被験体としては、例えば、特定の疾患に罹患している被験体、および特定の疾患に罹患している可能性がある被験体が挙げられる。特定の疾患としては、例えば、癌（例、肝臓癌、前立腺癌、悪性黒色腫）、肝臓疾患（例、肝炎、肝硬変）が挙げられる（例えば、国際公開第２００４／０３８４２０号；国際公開第２００７／０８１７９０号；国際公開第２００５／０３９３８０；Ｄｅｔｅｃｔｉｏｎ ｏｆ ｇｌｙｐｉｃａｎ－３－ｓｐｅｃｉｆｉｃ ＣＴＬｓ ｉｎ ｃｈｒｏｎｉｃ ｈｅｐａｔｉｔｉｓ ａｎｄ ｌｉｖｅｒ ｃｉｒｒｈｏｓｉｓ，Ｏｎｃｏｌｏｇｙ Ｒｅｐｏｒｔｓ ２２，ｐ．１４９－５４，２００９を参照）。
In a particular embodiment, the soluble GPC3 containing sample may be a sample obtained from a subject in a particular state. Examples of such a subject include a subject suffering from a specific disease and a subject who may have a specific disease. Specific diseases include, for example, cancer (eg, liver cancer, prostate cancer, malignant melanoma), liver disease (eg, hepatitis, cirrhosis) (eg, WO 2004/038420; WO 2007). / 081790; International Publication No. 2005/039380; Detection of glypican-3-specific CTLs in chronic hepatitis and liver cirrhosis, Oncology Reports 22, p. 149-54, p. 149-54.
可溶性ＧＰＣ３含有検体を還元剤と混合する場合、任意の還元剤を使用することができる。このような還元剤としては、例えば、２－（ジメチルアミノ）エタンチオール（ＤＥＡＥＴ）、トリス（２－カルボキシエチル）ホスフィン（ＴＣＥＰ）、２－メルカプトエチルアミン、２－メルカプトエタノール、ジチオトレイトール、チオグリセロール、亜硫酸ナトリウム、およびボロハイドライド、ならびにそれらの塩が挙げられる。塩としては、例えば、金属塩（例、ナトリウム塩、カリウム塩等の一価の金属塩、およびカルシウム塩、マグネシウム塩等の二価の金属塩）、無機塩（例、フッ化物、塩化物、臭化物、ヨウ化物等のハロゲン化物塩、およびアンモニウム塩）、有機塩（例、アルキル基で置換されたアンモニウム塩）、および酸付加塩（例、硫酸、塩酸、臭化水素酸、硝酸、リン酸等の無機酸との塩、および酢酸、シュウ酸、乳酸、クエン酸、トリフルオロメタンスルホン酸、トリフルオロ酢酸等の有機酸との塩）が挙げられる。溶液中で高度に安定である還元剤の使用等の観点では、ＤＥＡＥＴ、もしくはＴＣＥＰ、またはそれらの塩が好ましい。
When mixing the soluble GPC3 containing sample with the reducing agent, any reducing agent can be used. Examples of such reducing agents include 2- (dimethylamino) ethanethiol (DEAET), tris (2-carboxyethyl) phosphine (TCEP), 2-mercaptoethylamine, 2-mercaptoethanol, dithiothreitol, and thioglycerol. , Sodium sulfite, and borohydride, and salts thereof. Examples of the salt include metal salts (eg, monovalent metal salts such as sodium salt and potassium salt, and divalent metal salts such as calcium salt and magnesium salt) and inorganic salts (eg, fluoride, chloride, etc.). Bromide, halide salts such as iodide, and ammonium salts), organic salts (eg, ammonium salts substituted with alkyl groups), and acid addition salts (eg, sulfuric acid, hydrochloric acid, hydrobromic acid, nitrate, phosphoric acid). Salts with inorganic acids such as acetic acid, oxalic acid, lactic acid, citric acid, trifluoromethanesulfonic acid, salts with organic acids such as trifluoroacetic acid). From the standpoint of using a reducing agent that is highly stable in solution, DEAET, TCEP, or salts thereof are preferred.
還元剤は、陰性検体からの検出シグナル強度（例、カウント）を低減できる終濃度で用いることができる。終濃度とは、混合の際の濃度である。したがって、終濃度は、混合により生成する混合液中の濃度としても表現できる。このような終濃度は、例えば０．０５～１，０００ｍＭ、好ましくは０．５～５００ｍＭ、より好ましくは１～３００ｍＭ、さらにより好ましくは３～２００ｍＭである。
The reducing agent can be used at a final concentration that can reduce the detection signal intensity (eg, count) from negative samples. The final concentration is the concentration at the time of mixing. Therefore, the final concentration can also be expressed as the concentration in the mixed solution produced by mixing. Such a final concentration is, for example, 0.05 to 1,000 mM, preferably 0.5 to 500 mM, more preferably 1 to 300 mM, and even more preferably 3 to 200 mM.
可溶性ＧＰＣ３含有検体および還元剤の混合では、可溶性ＧＰＣ３含有検体を界面活性剤とさらに混合することを含んでいてもよい。このような場合、可溶性ＧＰＣ３含有検体、還元剤、および界面活性剤の混合液が生成する。
Mixing the soluble GPC3 containing sample and the reducing agent may include further mixing the soluble GPC3 containing sample with the surfactant. In such a case, a mixed solution of a soluble GPC3-containing sample, a reducing agent, and a surfactant is produced.
界面活性剤としては、例えば、非イオン性界面活性剤、両イオン性界面活性剤、陰イオン性界面活性剤、および陽イオン性界面活性剤、ならびにそれらの塩が挙げられる。塩は、上述のものと同様である。非イオン性界面活性剤としては、例えば、ポリオキシエチレンソルビタン脂肪酸エステル、ポリオキシエチレンアルキルフェニルエーテル、およびポリオキシエチレンアルキルエーテルを挙げることができる。ポリオキシエチレンソルビタン脂肪酸エステルとしては、例えば、ポリオキシエチレンソルビタンモノラウレート（Ｔｗｅｅｎ２０）、ポリオキシエチレンソルビタンモノパルミタート（Ｔｗｅｅｎ４０）、ポリオキシエチレンソルビタンモノステアレート（Ｔｗｅｅｎ６０）、ポリオキシエチレンソルビタンモノオレエート（Ｔｗｅｅｎ８０）が挙げられる。ポリオキシエチレンアルキルフェニルエーテルとしては、例えば、ポリオキシエチレン（１０）オクチルフェニルエーテル（Ｔｒｉｔｏｎ Ｘ－１００）、ポリオキシエチレン（８）オクチルフェニルエーテル（Ｔｒｉｔｏｎ Ｘ－１１４）、ポリオキシエチレン（３０）オクチルフェニルエーテル（Ｔｒｉｔｏｎ Ｘ－３０５）、およびポリオキシエチレン（４０）オクチルフェニルエーテル（Ｔｒｉｔｏｎ Ｘ－４０５）が挙げられる。ポリオキシエチレンアルキルエーテルとしては、ポリオキシエチレン（２３）ラウリルエーテル（Ｂｒｉｊ３５）、ポリオキシエチレン（２０）セチルエーテル（Ｂｒｉｊ５８）が挙げられる。非イオン性界面活性剤の好ましい例としては、ポリオキシエチレンソルビタンモノラウレート（Ｔｗｅｅｎ２０）、およびポリオキシエチレン（１０）オクチルフェニルエーテル（Ｔｒｉｔｏｎ Ｘ－１００）が挙げられる。両イオン性界面活性剤としては、例えば、スルホベタイン型界面活性剤が挙げられる。スルホベタイン型界面活性剤としては、例えば、３－［（３－コラミドプロピル）ジメチルアンモニオ］－１－プロパンスルホナート（ＣＨＡＰＳ）、３－［３－コラミドプロピル］ジメチルアンモニオ）－２－ヒドロキシプロパンスルホナート（ＣＨＡＰＳＯ）、Ｎ－ドデシル－Ｎ，Ｎ－ジメチル－３－アンモニオ－１－プロパンスルホネート、Ｎ－テトラデシル－Ｎ，Ｎ－ジメチル－３－アンモニオ－１－プロパンスルホネート、およびＮ－ヘキサデシル－Ｎ，Ｎ－ジメチル－３－アンモニオ－１－プロパンスルホネートが挙げられる。陰イオン性界面活性剤としては、例えば、ドデシル硫酸ナトリウム（ＳＤＳ）、Ｎ－ラウロイルサルコシンナトリウム（ＮＬＳ）ドデシル硫酸リチウム、ドデシルベンゼンスルホン酸ナトリウム、およびデオキシコール酸塩が挙げられる。陽イオン界面活性剤としては、例えば、デシルトリメチルアンモニウムクロライド、ドデシルトリメチルアンモニウムクロライド、テトラデシルトリメチルアンモニウムクロライド、ヘキサデシルトリメチルアンモニウムクロライド、デシルトリメチルアンモニウムブロマイド、ドデシルトリメチルアンモニウムブロマイド、テトラデシルトリメチルアンモニウムブロマイド、およびヘキサデシルトリメチルアンモニウムブロマイドが挙げられる。好ましくは、界面活性剤は、非イオン性界面活性剤、もしくは両イオン性界面活性剤、またはそれらの塩である。
Examples of the surfactant include nonionic surfactants, amphoteric surfactants, anionic surfactants, and cationic surfactants, and salts thereof. The salt is similar to that described above. Examples of the nonionic surfactant include polyoxyethylene sorbitan fatty acid ester, polyoxyethylene alkyl phenyl ether, and polyoxyethylene alkyl ether. Examples of the polyoxyethylene sorbitan fatty acid ester include polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monopalmitate (Tween 40), polyoxyethylene sorbitan monostearate (Tween 60), and polyoxyethylene sorbitan monoole. Ate (Tween 80) can be mentioned. Examples of the polyoxyethylene alkyl phenyl ether include polyoxyethylene (10) octylphenyl ether (Triton X-100), polyoxyethylene (8) octylphenyl ether (Triton X-114), and polyoxyethylene (30) octyl. Examples thereof include phenyl ether (Triton X-305) and polyoxyethylene (40) octyl phenyl ether (Triton X-405). Examples of the polyoxyethylene alkyl ether include polyoxyethylene (23) lauryl ether (Brij35) and polyoxyethylene (20) cetyl ether (Brij58). Preferred examples of the nonionic surfactant include polyoxyethylene sorbitan monolaurate (Tween 20) and polyoxyethylene (10) octylphenyl ether (Triton X-100). Examples of the zwitterionic surfactant include a sulfobetaine type surfactant. Examples of the sulfobetaine-type surfactant include 3-[(3-colamidpropyl) dimethylammonio] -1-propanesulfonate (CHAPS) and 3- [3-colamidpropyl] dimethylammonio) -2. -Hydroxypropanesulfonate (CHAPSO), N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, and N- Hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate can be mentioned. Anionic surfactants include, for example, sodium dodecyl sulfate (SDS), sodium N-lauroyl sarcosin (NLS) lithium dodecyl sulfate, sodium dodecylbenzene sulfonate, and deoxycholate. Examples of the cationic surfactant include decyltrimethylammonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride, decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, and hexa. Calcyltrimethylammonium bromide can be mentioned. Preferably, the surfactant is a nonionic surfactant, or an amphoteric surfactant, or a salt thereof.
界面活性剤は、陰性検体からの検出シグナル強度を低減する還元剤の効果を増強できる、および／または検体のマトリックス効果を低減できる終濃度で用いることができる。終濃度とは、混合の際の濃度である。したがって、終濃度は、混合により生成する混合液中の濃度としても表現できる。このような終濃度は、例えば０．００５～１０重量％、好ましくは０．０１～９重量％、より好ましくは０．０２～７．５重量％、さらにより好ましくは０．０５～６重量％、特に好ましくは０．０５～５重量％である。
The surfactant can be used at a final concentration that can enhance the effect of the reducing agent on reducing the detection signal intensity from the negative sample and / or reduce the matrix effect of the sample. The final concentration is the concentration at the time of mixing. Therefore, the final concentration can also be expressed as the concentration in the mixed solution produced by mixing. Such a final concentration is, for example, 0.005 to 10% by weight, preferably 0.01 to 9% by weight, more preferably 0.02 to 7.5% by weight, and even more preferably 0.05 to 6% by weight. , Particularly preferably 0.05 to 5% by weight.
好ましくは、還元剤および界面活性剤は、陰性検体からの検出シグナル強度の低減、および／または検体のマトリックス効果の低減において効果を増強することができる比率で用いることができる。このような比率は、還元剤１ｍＭあたりの界面活性剤の濃度範囲により規定することができる。還元剤１ｍＭあたりの界面活性剤の濃度範囲は、例えば０．００００２５～３重量％、好ましくは０．００００５～０．１重量％、より好ましくは０．０００５～０．０１重量％、さらにより好ましくは０．００５～０．０５重量％である。
Preferably, the reducing agent and the surfactant can be used in a ratio capable of enhancing the effect in reducing the detection signal intensity from the negative sample and / or reducing the matrix effect of the sample. Such a ratio can be defined by the concentration range of the surfactant per 1 mM of the reducing agent. The concentration range of the surfactant per 1 mM of the reducing agent is, for example, 0.000025 to 3% by weight, preferably 0.00005 to 0.1% by weight, more preferably 0.0005 to 0.01% by weight, and even more preferably. Is 0.005 to 0.05% by weight.
可溶性ＧＰＣ３含有検体が還元剤および界面活性剤の双方と混合される場合、混合は、同時または別々に行うことができる。混合が同時に行われる場合、可溶性ＧＰＣ３含有検体を、還元剤および界面活性剤の混合液と混合することができる。混合が別々に行われる場合、可溶性ＧＰＣ３含有検体を、先ず還元剤、次に界面活性剤と混合してもよく、または先ず界面活性剤、次に還元剤と混合してもよい。
When the soluble GPC3 containing sample is mixed with both the reducing agent and the surfactant, the mixing can be performed simultaneously or separately. When mixing is carried out simultaneously, the soluble GPC3 containing sample can be mixed with a mixture of reducing agent and surfactant. If the mixing is done separately, the soluble GPC3 containing sample may be mixed first with the reducing agent and then with the surfactant, or first with the surfactant and then with the reducing agent.
還元剤および／または界面活性剤は、水溶液中に溶解して用いることができる。このような水溶液としては、例えば、水（例、蒸留水、滅菌水、滅菌蒸留水、純水）、および緩衝液が挙げられる。緩衝液としては、例えば、リン酸緩衝液、ＭＥＳ緩衝液、クエン酸緩衝液、Ｔｒｉｓ緩衝液、炭酸緩衝液、ＨＰＥＰＳ緩衝液、ＭＯＰＳ緩衝液が挙げられる。緩衝液のｐＨは、還元剤の種類および濃度等の因子に応じて変動するが、還元剤の効果の向上等の観点から、１．０～９．０（好ましくは４．０～６．０）であってもよい。所望のｐＨ範囲で還元剤の効果を安定して発揮させる等の観点から、水溶液は、好ましくは緩衝液である。水溶液は、有機溶媒（例、アルコール）等の他の成分を含んでいてもよい。可溶性ＧＰＣ３含有検体と、還元剤および／または界面活性剤を含む水溶液とで混合されるべき容量の比率（可溶性ＧＰＣ３含有検体：還元剤および／または界面活性剤を含む水溶液）は、例えば１０：１～１：１０であり、好ましくは５：１～１：５であり、より好ましくは２：１～１：２である。
The reducing agent and / or surfactant can be used by dissolving it in an aqueous solution. Examples of such an aqueous solution include water (eg, distilled water, sterilized water, sterilized distilled water, pure water), and a buffer solution. Examples of the buffer solution include phosphate buffer solution, MES buffer solution, citric acid buffer solution, Tris buffer solution, carbon dioxide buffer solution, HPEPS buffer solution, and MOPS buffer solution. The pH of the buffer solution varies depending on factors such as the type and concentration of the reducing agent, but from the viewpoint of improving the effect of the reducing agent, it is 1.0 to 9.0 (preferably 4.0 to 6.0). ) May be. The aqueous solution is preferably a buffer solution from the viewpoint of stably exerting the effect of the reducing agent in a desired pH range. The aqueous solution may contain other components such as an organic solvent (eg, alcohol). The ratio of the volume to be mixed with the soluble GPC3-containing sample and the aqueous solution containing the reducing agent and / or the surfactant (soluble GPC3-containing sample: the aqueous solution containing the reducing agent and / or the surfactant) is, for example, 10: 1. It is ~ 1:10, preferably 5: 1 to 1: 5, and more preferably 2: 1 to 1: 2.
混合は、還元剤単独、または還元剤および界面活性剤の双方による検体の処理に十分な条件下で行われる。このような温度条件は、例えば１５～６０℃、好ましくは２０～５０℃、より好ましくは２５～４５℃である。混合時間は、例えば、３０秒以下である。迅速な処理等の観点から、混合時間は、好ましくは２０秒以下であり、より好ましくは１５秒以下である。このような条件下で混合することにより、陰性検体からの検出シグナル強度を低減することができる。
Mixing is carried out under conditions sufficient for processing the sample with the reducing agent alone or with both the reducing agent and the surfactant. Such temperature conditions are, for example, 15 to 60 ° C., preferably 20 to 50 ° C., more preferably 25 to 45 ° C. The mixing time is, for example, 30 seconds or less. From the viewpoint of rapid processing and the like, the mixing time is preferably 20 seconds or less, more preferably 15 seconds or less. By mixing under such conditions, the intensity of the detection signal from the negative sample can be reduced.
本発明の方法は、混合後に、さらに混合液をインキュベートすることを含んでいてもよい。インキュベート時間は、還元剤の種類および濃度、界面活性剤の併用の有無、界面活性剤の種類および濃度、混合時間、および検出シグナル強度において所望される低減度、ならびに抗体を用いた測定（イムノアッセイ）が行われる場合には、それに要する時間等の因子に応じて変動するが、例えば１２０分以下、好ましくは６０分以下、より好ましくは３０分以下である。迅速な処理等の観点から、インキュベート時間は、さらにより好ましくは２０分以下であり、特に好ましくは１０分以下、または５分以下であってもよい。インキュベート温度は、上述の混合における温度条件と同様である。
The method of the present invention may include further incubating the mixed solution after mixing. Incubation time is determined by the type and concentration of reducing agent, the presence or absence of a surfactant in combination, the type and concentration of surfactant, the mixing time, and the desired reduction in detection signal intensity, and measurement using an antibody (immunoassay). When is performed, it varies depending on factors such as the time required for it, but is, for example, 120 minutes or less, preferably 60 minutes or less, and more preferably 30 minutes or less. From the viewpoint of rapid treatment and the like, the incubation time is even more preferably 20 minutes or less, and particularly preferably 10 minutes or less, or 5 minutes or less. The incubation temperature is similar to the temperature conditions in the mixing described above.
本発明はまた、下記（ａ）および（ｂ）を含む、可溶性ＧＰＣ３のイムノアッセイ方法を提供する：
（ａ）可溶性ＧＰＣ３含有検体を還元剤と混合すること；および
（ｂ）（ａ）で得られた混合液中の可溶性ＧＰＣ３量を、可溶性ＧＰＣ３に対する１種以上の抗体を用いて測定すること。 The present invention also provides an immunoassay method for soluble GPC3, which comprises the following (a) and (b):
(A) Mixing a soluble GPC3-containing sample with a reducing agent; and (b) measuring the amount of soluble GPC3 in the mixture obtained in (a) using one or more antibodies against soluble GPC3.
（ａ）可溶性ＧＰＣ３含有検体を還元剤と混合すること；および
（ｂ）（ａ）で得られた混合液中の可溶性ＧＰＣ３量を、可溶性ＧＰＣ３に対する１種以上の抗体を用いて測定すること。 The present invention also provides an immunoassay method for soluble GPC3, which comprises the following (a) and (b):
(A) Mixing a soluble GPC3-containing sample with a reducing agent; and (b) measuring the amount of soluble GPC3 in the mixture obtained in (a) using one or more antibodies against soluble GPC3.
可溶性ＧＰＣ３のイムノアッセイ方法における各種要素の定義、例、および好ましい例は、本発明による可溶性ＧＰＣ３含有検体の処理方法において述べたものと同様である。
Definitions, examples, and preferred examples of various elements in the method for immunoassaying soluble GPC3 are the same as those described in the method for treating soluble GPC3-containing samples according to the present invention.
工程（ａ）は、本発明による可溶性ＧＰＣ３含有検体の処理方法と同様にして行うことができる。工程（ｂ）は、上述のイムノアッセイにより行うことができる。工程（ａ）および（ｂ）は、並行してまたは別々に行うことができる。例えば、工程（ａ）および（ｂ）が並行して行われる場合、可溶性ＧＰＣ３含有検体、還元剤（および界面活性剤）、ならびに可溶性ＧＰＣ３に対する１種以上の抗体を同時に混合することにより、還元剤（および界面活性剤）による可溶性ＧＰＣ３含有検体の処理と抗原抗体反応を同時に行うことができる。しかし、陰性検体からの検出シグナル強度（例、カウント）を十分に低減させるためには、還元剤（および界面活性剤）による可溶性ＧＰＣ３含有検体の処理を十分に行い、次いで、可溶性ＧＰＣ３に対する１種以上の抗体を用いた抗原抗体反応により可溶性ＧＰＣ３量を測定することが好ましい。また、本発明で用いられる抗体がジスルフィド結合を含む場合、抗体と還元剤を長時間共存させると、還元剤は、抗体中のジスルフィド結合の切断により抗体を破壊するので、イムノアッセイの測定精度に影響し得る。したがって、本発明で用いられる抗体がジスルフィド結合を含む場合、工程（ａ）および（ｂ）は、別々に行われることが好ましい。勿論、本発明で用いられる抗体がジスルフィド結合を含まない場合（例、単鎖抗体）、工程（ａ）および（ｂ）を並行して行うこともまた好ましい。
Step (a) can be performed in the same manner as the method for treating a soluble GPC3-containing sample according to the present invention. Step (b) can be performed by the immunoassay described above. Steps (a) and (b) can be performed in parallel or separately. For example, when steps (a) and (b) are performed in parallel, a reducing agent can be obtained by simultaneously mixing a soluble GPC3 containing sample, a reducing agent (and a surfactant), and one or more antibodies against the soluble GPC3. Treatment of the soluble GPC3-containing sample with (and surfactant) and antigen-antibody reaction can be performed simultaneously. However, in order to sufficiently reduce the detection signal intensity (eg, count) from negative samples, the soluble GPC3-containing sample should be sufficiently treated with a reducing agent (and surfactant), and then one of the soluble GPC3-containing samples. It is preferable to measure the amount of soluble GPC3 by an antigen-antibody reaction using the above antibody. Further, when the antibody used in the present invention contains a disulfide bond, if the antibody and the reducing agent coexist for a long time, the reducing agent destroys the antibody by cleaving the disulfide bond in the antibody, which affects the measurement accuracy of the immunoassay. Can be. Therefore, when the antibody used in the present invention contains a disulfide bond, steps (a) and (b) are preferably performed separately. Of course, when the antibody used in the present invention does not contain a disulfide bond (eg, single chain antibody), it is also preferable to carry out steps (a) and (b) in parallel.
本発明はさらに、下記（ａ）および（ｂ）を含む、可溶性ＧＰＣ３のイムノアッセイ試薬を提供する：
（ａ）還元剤；および
（ｂ）可溶性ＧＰＣ３に対する１種以上の抗体。 The present invention further provides an immunoassay reagent for soluble GPC3, which comprises the following (a) and (b):
(A) Reducing agent; and (b) One or more antibodies against soluble GPC3.
（ａ）還元剤；および
（ｂ）可溶性ＧＰＣ３に対する１種以上の抗体。 The present invention further provides an immunoassay reagent for soluble GPC3, which comprises the following (a) and (b):
(A) Reducing agent; and (b) One or more antibodies against soluble GPC3.
本発明の試薬は、さらに（ｃ）界面活性剤を含んでいてもよい。
The reagent of the present invention may further contain (c) a surfactant.
構成成分（ａ）～（ｃ）の還元剤、抗体、および界面活性剤の定義、例、および好ましい例は、本発明による可溶性ＧＰＣ３含有検体の処理方法において述べたものと同様である。
Definitions, examples, and preferred examples of the reducing agents, antibodies, and surfactants of the constituents (a) to (c) are the same as those described in the method for treating a soluble GPC3-containing sample according to the present invention.
特定の実施形態では、本発明の試薬は、可溶性ＧＰＣ３に対する１種以上の抗体が、可溶性ＧＰＣ３中の異なる２種以上のエピトープに対する異なる２種以上の抗体であり、かつ、前記試薬がサンドイッチイムノアッセイ用である試薬であってもよい。好ましくは、このような試薬は、上記２種以上の抗体として、可溶性ＧＰＣ３に対する標識抗体および／または固相抗体を含む。あるいは、このような試薬は、標識物質で標識された標識抗体および／または固相に固定された固相抗体を含まない場合、標識物質および／または固相を含んでいてもよい。標識物質としては、例えば、蛍光物質、発光物質、色素、および酵素が挙げられる。標識物質が酵素である場合、このような試薬は、酵素の基質（例、検出シグナルを発生する基質、または酵素により検出シグナルを発生する生成物に変換される基質、あるいは、検出シグナルを発生する基質、または酵素により検出シグナルを発生する生成物に変換される基質を利用する他の酵素反応と共役し得る反応における基質）を含んでいてもよい。固相は、上述したものと同様である。
In certain embodiments, the reagents of the invention are such that one or more antibodies against soluble GPC3 are two or more different antibodies against two or more different epitopes in soluble GPC3, and the reagents are for sandwich immunoassays. It may be a reagent that is. Preferably, such reagents include labeled and / or solid phase antibodies against soluble GPC3 as the two or more antibodies described above. Alternatively, such reagents may include a labeling substance and / or a solid phase if it does not contain a labeling antibody labeled with a labeling substance and / or a solid phase antibody immobilized on a solid phase. Labeling substances include, for example, fluorescent substances, luminescent substances, dyes, and enzymes. When the labeling substance is an enzyme, such a reagent produces a substrate of the enzyme (eg, a substrate that produces a detection signal, or a substrate that is converted by the enzyme into a product that produces a detection signal, or a detection signal. It may also include a substrate, or a substrate in a reaction that can be coupled to another enzymatic reaction that utilizes a substrate that is converted into a product that produces a detection signal by the enzyme. The solid phase is similar to that described above.
本発明の試薬は、特定の状態の判定（例、疾患の診断）に用いることができる。特定の状態（例、疾患）は、上述したものと同様である。好ましくは、本発明の試薬は、肝臓癌、前立腺癌等の癌の診断に用いることができる。
The reagent of the present invention can be used for determining a specific state (eg, diagnosing a disease). Specific conditions (eg, disease) are similar to those described above. Preferably, the reagent of the present invention can be used for diagnosing cancers such as liver cancer and prostate cancer.
以下、実施例を参照して本発明をより詳細に説明するが、本発明は、これらの実施例に限定されるものではない。
Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.
参考例１：グリピカン－３（ＧＰＣ３）と抗体の反応性の確認
ＧＰＣ３の１－５５９番目のアミノ酸残基からなる組み換えヒトＧＰＣ３（ｒｈＧＰＣ３）（Ｒ＆Ｄ Ｓｙｓｔｅｍｓ）抗原、またはＧＰＣ３高発現のヒト肝癌細胞ＨｅｐＧ２の培養上清を用いたウェスタンブロッティングにより、抗ＧＰＣ３抗体Ａおよび抗ＧＰＣ３抗体Ｂ（いずれもモノクローナルＩｇＧ抗体）の認識部位の解析を行った。 Reference Example 1: Confirmation of antibody reactivity with glypican-3 (GPC3) Recombinant human GPC3 (rhGPC3) (R & D Systems) antigen consisting of amino acid residue at position 1-559 of GPC3, or human hepatoma cell HepG2 with high expression of GPC3 The recognition sites of anti-GPC3 antibody A and anti-GPC3 antibody B (both monoclonal IgG antibodies) were analyzed by Western blotting using the culture supernatant of.
ＧＰＣ３の１－５５９番目のアミノ酸残基からなる組み換えヒトＧＰＣ３（ｒｈＧＰＣ３）（Ｒ＆Ｄ Ｓｙｓｔｅｍｓ）抗原、またはＧＰＣ３高発現のヒト肝癌細胞ＨｅｐＧ２の培養上清を用いたウェスタンブロッティングにより、抗ＧＰＣ３抗体Ａおよび抗ＧＰＣ３抗体Ｂ（いずれもモノクローナルＩｇＧ抗体）の認識部位の解析を行った。 Reference Example 1: Confirmation of antibody reactivity with glypican-3 (GPC3) Recombinant human GPC3 (rhGPC3) (R & D Systems) antigen consisting of amino acid residue at position 1-559 of GPC3, or human hepatoma cell HepG2 with high expression of GPC3 The recognition sites of anti-GPC3 antibody A and anti-GPC3 antibody B (both monoclonal IgG antibodies) were analyzed by Western blotting using the culture supernatant of.
ｒｈＧＰＣ３とＨｅｐＧ２培養上清にＳＤＳ－ＰＡＧＥ用サンプルバッファーを添加し、それぞれ１．４ｎｇタンパク質／レーンと７．２ｎｇタンパク質／レーンとなるようにｒｈＧＰＣ３とＨｅｐＧ２培養上清を５－２０％のポリアクリルアミドゲル（スーパーセットエース５－２０％、ＷＡＫＯ）にアプライした。電気泳動（３０ｍＡ、６０分）後、トランスブロット（登録商標）ＳＤセミドライ電気泳動転写セル（Ｂｉｏ－Ｒａｄ）を用いてブロッティングメンブレン（イモビロンＩＳＥＱ、メルクミリポア）へタンパク質を転写した（１５Ｖ、６０分）。ブロッティングメンブレンを、ＴＢＳ－Ｔで軽く洗った後、ブロッキング液（０．５％ＥＣＬ Ｂｌоｃｋ（ＧＥヘルスケア ライフサイエンス）、０．５％ＢＳＡ、ＴＢＳ－Ｔ）中で１時間、室温で振とうした。ＴＢＳ－Ｔで２回洗浄後、ブロッティングメンブレンを、反応液（ブロッキング液をＴＢＳ－Ｔで２倍希釈）で１μｇ／ｍＬに各々希釈した抗ＧＰＣ３抗体Ａと抗ＧＰＣ３抗体Ｂとを含む溶液中で４℃で一晩振とうした。ＴＢＳ－Ｔで４回洗浄後、ブロッティングメンブレンを、反応液で２０，０００倍希釈したＰＯＤ標識抗マウス Ｆ（ａｂ’）２（Ｊａｃｋｓｏｎ）を含む溶液中で１時間振とう後、ＴＢＳ－Ｔで洗浄した。発色反応はＳｕｐｅｒＳｉｇｎａｌ Ｗｅｓｔ Ｆｅｍｔｏ Ｍａｘｉｍｕｍ Ｓｅｎｓｉｔｉｖｉｔｙ Ｓｕｂｓｔｒａｔｅ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ）を用いて行い、ケミルミイメージングシステム（ＦＵＳＩＯＮ ＳＹＳＴＥＭ、ビルバー・ルーマット）を用いて現像した。ウェスタンブロッティングの解析結果を図１に示す。
その結果、抗ＧＰＣ３抗体Ａと抗ＧＰＣ３抗体Ｂはいずれも、ＧＰＣ３のＮ末端断片（３５８番目のアミノ酸残基よりもＮ末端側の断片）に相当する４０ｋＤａのバンドに反応した（図１）。このことは、抗ＧＰＣ３抗体Ａおよび抗ＧＰＣ３抗体Ｂは、ＧＰＣ３のＮ末端領域を認識することを示す。したがって、抗ＧＰＣ３抗体Ａおよび抗ＧＰＣ３抗体Ｂが可溶性ＧＰＣ３を認識することが確認された。 A sample buffer for SDS-PAGE was added to the rhGPC3 and HepG2 culture supernatants, and the rhGPC3 and HepG2 culture supernatants were mixed with a 5-20% polyacrylamide gel so as to have 1.4 ng protein / lane and 7.2 ng protein / lane, respectively. Applied to (Superset Ace 5-20%, WAKO). After electrophoresis (30 mA, 60 minutes), the protein was transferred to a blotting membrane (Imoviron ISEC, Merck Millipore) using a Transblot® SD semi-dry electrophoresis transfer cell (Bio-Rad) (15 V, 60 minutes). .. The blotting membrane was lightly washed with TBS-T and then shaken in a blocking solution (0.5% ECL Blоck (GE Healthcare Life Science), 0.5% BSA, TBS-T) for 1 hour at room temperature. .. After washing twice with TBS-T, the blotting membrane was diluted in 1 μg / mL with a reaction solution (blocking solution diluted 2-fold with TBS-T) in a solution containing anti-GPC3 antibody A and anti-GPC3 antibody B, respectively. Shake overnight at 4 ° C. After washing 4 times with TBS-T, the blotting membrane is shaken in a solution containing POD-labeled anti-mouse F (ab') 2 (Jackson) diluted 20,000 times with the reaction solution for 1 hour, and then with TBS-T. Washed. The color development reaction was carried out using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and developed using a Chemilmi imaging system (FUSION SYSTEM, Biller Lumat). The analysis result of Western blotting is shown in FIG.
As a result, both anti-GPC3 antibody A and anti-GPC3 antibody B reacted with a band of 40 kDa corresponding to the N-terminal fragment of GPC3 (the fragment on the N-terminal side of the 358th amino acid residue) (FIG. 1). This indicates that anti-GPC3 antibody A and anti-GPC3 antibody B recognize the N-terminal region of GPC3. Therefore, it was confirmed that anti-GPC3 antibody A and anti-GPC3 antibody B recognize soluble GPC3.
その結果、抗ＧＰＣ３抗体Ａと抗ＧＰＣ３抗体Ｂはいずれも、ＧＰＣ３のＮ末端断片（３５８番目のアミノ酸残基よりもＮ末端側の断片）に相当する４０ｋＤａのバンドに反応した（図１）。このことは、抗ＧＰＣ３抗体Ａおよび抗ＧＰＣ３抗体Ｂは、ＧＰＣ３のＮ末端領域を認識することを示す。したがって、抗ＧＰＣ３抗体Ａおよび抗ＧＰＣ３抗体Ｂが可溶性ＧＰＣ３を認識することが確認された。 A sample buffer for SDS-PAGE was added to the rhGPC3 and HepG2 culture supernatants, and the rhGPC3 and HepG2 culture supernatants were mixed with a 5-20% polyacrylamide gel so as to have 1.4 ng protein / lane and 7.2 ng protein / lane, respectively. Applied to (Superset Ace 5-20%, WAKO). After electrophoresis (30 mA, 60 minutes), the protein was transferred to a blotting membrane (Imoviron ISEC, Merck Millipore) using a Transblot® SD semi-dry electrophoresis transfer cell (Bio-Rad) (15 V, 60 minutes). .. The blotting membrane was lightly washed with TBS-T and then shaken in a blocking solution (0.5% ECL Blоck (GE Healthcare Life Science), 0.5% BSA, TBS-T) for 1 hour at room temperature. .. After washing twice with TBS-T, the blotting membrane was diluted in 1 μg / mL with a reaction solution (blocking solution diluted 2-fold with TBS-T) in a solution containing anti-GPC3 antibody A and anti-GPC3 antibody B, respectively. Shake overnight at 4 ° C. After washing 4 times with TBS-T, the blotting membrane is shaken in a solution containing POD-labeled anti-mouse F (ab') 2 (Jackson) diluted 20,000 times with the reaction solution for 1 hour, and then with TBS-T. Washed. The color development reaction was carried out using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and developed using a Chemilmi imaging system (FUSION SYSTEM, Biller Lumat). The analysis result of Western blotting is shown in FIG.
As a result, both anti-GPC3 antibody A and anti-GPC3 antibody B reacted with a band of 40 kDa corresponding to the N-terminal fragment of GPC3 (the fragment on the N-terminal side of the 358th amino acid residue) (FIG. 1). This indicates that anti-GPC3 antibody A and anti-GPC3 antibody B recognize the N-terminal region of GPC3. Therefore, it was confirmed that anti-GPC3 antibody A and anti-GPC3 antibody B recognize soluble GPC3.
参考例２：抗ＧＰＣ３抗体固相化粒子の調製
１０ｍＭ ＭＥＳ緩衝液（ｐＨ５．０）中で磁性粒子に抗ＧＰＣ３抗体Ａを添加して、０．２ｍｇ／ｍＬ 抗ＧＰＣ３抗体Ａおよび０．０１ｇ／ｍＬ 磁性粒子を含む懸濁液を得た。この懸濁液をゆるやかに攪拌しながら５℃で１時間インキュベートして、抗ＧＰＣ３抗体Ａを磁性粒子に固相化した。その後、磁性粒子を磁石で集磁し、磁性粒子を洗浄液（５０ｍＭ トリス緩衝液、１５０ｍＭ ＮａＣｌ、２．０％ＢＳＡ、ｐＨ７．２）にて洗浄し、抗ＧＰＣ３抗体Ａ固相化粒子を得た。測定では、抗ＧＰＣ３抗体Ａ固相化粒子を、粒子希釈液（５０ｍＭ Ｔｒｉｓ緩衝液、１ｍＭ ＥＤＴＡ２Ｎａ、０．１％ ＮａＮ３、２．０％ＢＳＡ、ｐＨ７．２）中に懸濁した。 Reference Example 2: Preparation of anti-GPC3 antibody-immobilized particles Anti-GPC3 antibody A is added to magnetic particles in 10 mM MES buffer (pH 5.0), and 0.2 mg / mL anti-GPC3 antibody A and 0.01 g / A suspension containing mL magnetic particles was obtained. The suspension was incubated at 5 ° C. for 1 hour with gentle stirring to solidify the anti-GPC3 antibody A into magnetic particles. Then, the magnetic particles were magnetized with a magnet, and the magnetic particles were washed with a washing solution (50 mM Tris buffer, 150 mM NaCl, 2.0% BSA, pH 7.2) to obtain anti-GPC3 antibody A immobilized particles. .. In the measurement, anti-GPC3 antibody A-immobilized particles were suspended in particle diluent (50 mM Tris buffer, 1 mM EDTA2Na, 0.1% NaN 3 , 2.0% BSA, pH 7.2).
１０ｍＭ ＭＥＳ緩衝液（ｐＨ５．０）中で磁性粒子に抗ＧＰＣ３抗体Ａを添加して、０．２ｍｇ／ｍＬ 抗ＧＰＣ３抗体Ａおよび０．０１ｇ／ｍＬ 磁性粒子を含む懸濁液を得た。この懸濁液をゆるやかに攪拌しながら５℃で１時間インキュベートして、抗ＧＰＣ３抗体Ａを磁性粒子に固相化した。その後、磁性粒子を磁石で集磁し、磁性粒子を洗浄液（５０ｍＭ トリス緩衝液、１５０ｍＭ ＮａＣｌ、２．０％ＢＳＡ、ｐＨ７．２）にて洗浄し、抗ＧＰＣ３抗体Ａ固相化粒子を得た。測定では、抗ＧＰＣ３抗体Ａ固相化粒子を、粒子希釈液（５０ｍＭ Ｔｒｉｓ緩衝液、１ｍＭ ＥＤＴＡ２Ｎａ、０．１％ ＮａＮ３、２．０％ＢＳＡ、ｐＨ７．２）中に懸濁した。 Reference Example 2: Preparation of anti-GPC3 antibody-immobilized particles Anti-GPC3 antibody A is added to magnetic particles in 10 mM MES buffer (pH 5.0), and 0.2 mg / mL anti-GPC3 antibody A and 0.01 g / A suspension containing mL magnetic particles was obtained. The suspension was incubated at 5 ° C. for 1 hour with gentle stirring to solidify the anti-GPC3 antibody A into magnetic particles. Then, the magnetic particles were magnetized with a magnet, and the magnetic particles were washed with a washing solution (50 mM Tris buffer, 150 mM NaCl, 2.0% BSA, pH 7.2) to obtain anti-GPC3 antibody A immobilized particles. .. In the measurement, anti-GPC3 antibody A-immobilized particles were suspended in particle diluent (50 mM Tris buffer, 1 mM EDTA2Na, 0.1% NaN 3 , 2.0% BSA, pH 7.2).
参考例３：アルカリホスファターゼ標識抗ＧＰＣ３抗体の調製
脱塩したアルカリホスファターゼ（ＡＬＰ）とＮ－（４－マレイミドブチリロキシ）－スクシンイミド（ＧＭＢＳ）（終濃度０．３ｍｇ／ｍＬ）を混合し、３０℃で１時間静置して、ＡＬＰをマレイミド化した。次いで、カップリング用反応液（１００ｍＭリン酸緩衝液、１ｍＭ ＥＤＴＡ２Ｎａ、ｐＨ６．３）中で、Ｆａｂ’化した抗ＧＰＣ３抗体Ｂと、マレイミド化ＡＬＰを１：１のモル比で混合し、２５℃で１時間反応させた。Ｓｕｐｅｒｄｅｘ２００ １０／３００（ＧＥ Ｈｅａｌｔｈｃａｒｅ）のカラムクロマトグラフィーを用いて、精製用緩衝液（５０ｍＭ ＭＥＳ緩衝液、１５０ｍＭ ＮａＣｌ、０．１％ＮａＮ３、ｐＨ８．０）で、流速０．５ｍＬ／ｍｉｎで主要ピークを分取して精製し、ＡＬＰ標識抗ＧＰＣ３抗体Ｂを得た。測定では、ＡＬＰ標識抗ＧＰＣ３抗体Ｂを標識体希釈液（５０ｍＭ ＭＥＳ緩衝液、１５０ｍＭ ＮａＣｌ、０．３ｍＭ ＺｎＣｌ２、１ｍＭ ＭｇＣｌ２、０．１％ ＮａＮ３、２．０％ ＢＳＡ、ｐＨ６．８）中に懸濁した。 Reference Example 3: Preparation of alkaline phosphatase-labeled anti-GPC3 antibody Desalted alkaline phosphatase (ALP) and N- (4-maleimide butyryloxy) -succinimide (GMBS) (final concentration 0.3 mg / mL) are mixed and 30 ° C. The ALP was maleimided by allowing it to stand for 1 hour. Next, Fab'formed anti-GPC3 antibody B and maleimided ALP were mixed in a coupling reaction solution (100 mM phosphate buffer, 1 mM EDTA2Na, pH 6.3) at a molar ratio of 1: 1 to 25 ° C. Was reacted for 1 hour. Mainly in purification buffer (50 mM MES buffer, 150 mM NaCl, 0.1% NaN 3 , pH 8.0) using column chromatography ofSuperdex200 10/300 (GE Healthcare) at a flow rate of 0.5 mL / min. The peak was separated and purified to obtain ALP-labeled anti-GPC3 antibody B. In the measurement, ALP-labeled anti-GPC3 antibody B was labeled as a diluent (50 mM MES buffer, 150 mM NaCl, 0.3 mM ZnCl 2 , 1 mM MgCl 2 , 0.1% NaN 3 , 2.0% BSA, pH 6.8). Suspended in.
脱塩したアルカリホスファターゼ（ＡＬＰ）とＮ－（４－マレイミドブチリロキシ）－スクシンイミド（ＧＭＢＳ）（終濃度０．３ｍｇ／ｍＬ）を混合し、３０℃で１時間静置して、ＡＬＰをマレイミド化した。次いで、カップリング用反応液（１００ｍＭリン酸緩衝液、１ｍＭ ＥＤＴＡ２Ｎａ、ｐＨ６．３）中で、Ｆａｂ’化した抗ＧＰＣ３抗体Ｂと、マレイミド化ＡＬＰを１：１のモル比で混合し、２５℃で１時間反応させた。Ｓｕｐｅｒｄｅｘ２００ １０／３００（ＧＥ Ｈｅａｌｔｈｃａｒｅ）のカラムクロマトグラフィーを用いて、精製用緩衝液（５０ｍＭ ＭＥＳ緩衝液、１５０ｍＭ ＮａＣｌ、０．１％ＮａＮ３、ｐＨ８．０）で、流速０．５ｍＬ／ｍｉｎで主要ピークを分取して精製し、ＡＬＰ標識抗ＧＰＣ３抗体Ｂを得た。測定では、ＡＬＰ標識抗ＧＰＣ３抗体Ｂを標識体希釈液（５０ｍＭ ＭＥＳ緩衝液、１５０ｍＭ ＮａＣｌ、０．３ｍＭ ＺｎＣｌ２、１ｍＭ ＭｇＣｌ２、０．１％ ＮａＮ３、２．０％ ＢＳＡ、ｐＨ６．８）中に懸濁した。 Reference Example 3: Preparation of alkaline phosphatase-labeled anti-GPC3 antibody Desalted alkaline phosphatase (ALP) and N- (4-maleimide butyryloxy) -succinimide (GMBS) (final concentration 0.3 mg / mL) are mixed and 30 ° C. The ALP was maleimided by allowing it to stand for 1 hour. Next, Fab'formed anti-GPC3 antibody B and maleimided ALP were mixed in a coupling reaction solution (100 mM phosphate buffer, 1 mM EDTA2Na, pH 6.3) at a molar ratio of 1: 1 to 25 ° C. Was reacted for 1 hour. Mainly in purification buffer (50 mM MES buffer, 150 mM NaCl, 0.1% NaN 3 , pH 8.0) using column chromatography of
参考例４：可溶性ＧＰＣ３の測定
前処理液２０μＬを反応槽に分注し、次に検体２０μＬを反応槽に分注した。前処理液と検体の混合液を、３７℃で６．５分間インキュベーションした後、反応槽に抗ＧＰＣ３抗体Ａ固相化粒子５０μＬを分注し、混合液を攪拌した。混合液を３７℃で８分間インキュベーションし、Ｂ／Ｆ分離・洗浄を行った。ＡＬＰ標識抗ＧＰＣ３抗体Ｂ ５０μＬを反応槽に分注した後、混合液を攪拌した。混合液を３７℃で８分間インキュベーションし、Ｂ／Ｆ分離・洗浄を行った。その後、化学発光基質である３－（２’－スピロアダマンタン）－４－メトキシ－４－（３’’－ホスホリルオキシ）フェニル－１，２－ジオキセタン・２ナトリウム塩（ＡＭＰＰＤ）を含むルミパルス基質液２００μＬを反応槽に分注し、混合液を攪拌した。その後、混合液を３７℃で４分間インキュベーションし、次に、発光量をルミノメーターで測定した。実際の測定は、全自動化学発光酵素免疫測定システム（ルミパルスＬ２４００（富士レビオ社製））にて行った。 Reference Example 4: Measurement ofsoluble GPC3 20 μL of the pretreatment solution was dispensed into the reaction vessel, and then 20 μL of the sample was dispensed into the reaction vessel. After incubating the mixed solution of the pretreatment solution and the sample at 37 ° C. for 6.5 minutes, 50 μL of anti-GPC3 antibody A-immobilized particles were dispensed into the reaction vessel, and the mixed solution was stirred. The mixed solution was incubated at 37 ° C. for 8 minutes to perform B / F separation and washing. After dispensing 50 μL of ALP-labeled anti-GPC3 antibody B into the reaction vessel, the mixed solution was stirred. The mixed solution was incubated at 37 ° C. for 8 minutes to perform B / F separation and washing. Then, a Lumipulse substrate solution containing a chemiluminescent substrate 3- (2'-spirodamantan) -4-methoxy-4- (3 ″ -phosphoryloxy) phenyl-1,2-dioxetane / 2-sodium salt (AMPPD). 200 μL was dispensed into the reaction vessel and the mixed solution was stirred. Then, the mixed solution was incubated at 37 ° C. for 4 minutes, and then the amount of luminescence was measured with a luminometer. The actual measurement was performed by a fully automated chemiluminescent enzyme immunoassay system (Lumipulse L2400 (manufactured by Fujirebio)).
前処理液２０μＬを反応槽に分注し、次に検体２０μＬを反応槽に分注した。前処理液と検体の混合液を、３７℃で６．５分間インキュベーションした後、反応槽に抗ＧＰＣ３抗体Ａ固相化粒子５０μＬを分注し、混合液を攪拌した。混合液を３７℃で８分間インキュベーションし、Ｂ／Ｆ分離・洗浄を行った。ＡＬＰ標識抗ＧＰＣ３抗体Ｂ ５０μＬを反応槽に分注した後、混合液を攪拌した。混合液を３７℃で８分間インキュベーションし、Ｂ／Ｆ分離・洗浄を行った。その後、化学発光基質である３－（２’－スピロアダマンタン）－４－メトキシ－４－（３’’－ホスホリルオキシ）フェニル－１，２－ジオキセタン・２ナトリウム塩（ＡＭＰＰＤ）を含むルミパルス基質液２００μＬを反応槽に分注し、混合液を攪拌した。その後、混合液を３７℃で４分間インキュベーションし、次に、発光量をルミノメーターで測定した。実際の測定は、全自動化学発光酵素免疫測定システム（ルミパルスＬ２４００（富士レビオ社製））にて行った。 Reference Example 4: Measurement of
実施例１：可溶性ＧＰＣ３の測定における還元剤ＤＥＡＥＴによる検体の前処理
健常人由来の血清検体（陰性検体）、およびα－フェトプロテイン（ＡＦＰ）陽性である肝臓がん患者由来の血清検体（Ｔｒｉｎａ社）（陽性検体）を、可溶性ＧＰＣ３評価用検体として使用した。前処理液として、６．２５ｍＭ～６００ｍＭの２－（ジメチルアミノ）エタンチオール塩酸塩（ＤＥＡＥＴ）を含むリン酸緩衝液（１０ｍＭリン酸緩衝液，６．２５～６００ｍＭ ＤＥＡＥＴ，ｐＨ６．０）を用いた。コントロール液として、１０ｍＭリン酸緩衝液（ｐＨ６．０）を用いた。陰性検体、陽性検体および緩衝液サンプル（１０ｍＭリン酸緩衝液）を、それぞれ、前処理液と体積比１：１で混合し、３７℃で６．５分間反応させた（前処理あり）。同様に、陰性検体、陽性検体および緩衝液サンプルを、それぞれ、コントロール液と混合し、反応させた（前処理なし）。 Example 1: Pretreatment of a sample with a reducing agent DEAET in the measurement of soluble GPC3 A serum sample derived from a healthy person (negative sample) and a serum sample derived from a liver cancer patient positive for α-fetoprotein (AFP) (Trina) (Positive sample) was used as a sample for evaluation of soluble GPC3. As the pretreatment solution, a phosphate buffer solution (10 mM phosphate buffer solution, 6.25-600 mM DEAET, pH 6.0) containing 6.25 mM to 600 mM 2- (dimethylamino) ethanethiol hydrochloride (DEAET) was used. board. As a control solution, a 10 mM phosphate buffer solution (pH 6.0) was used. Negative sample, positive sample and buffer sample (10 mM phosphate buffer) were mixed with the pretreatment solution at a volume ratio of 1: 1 and reacted at 37 ° C. for 6.5 minutes (with pretreatment). Similarly, the negative sample, the positive sample and the buffer solution sample were mixed with the control solution and reacted (without pretreatment).
健常人由来の血清検体（陰性検体）、およびα－フェトプロテイン（ＡＦＰ）陽性である肝臓がん患者由来の血清検体（Ｔｒｉｎａ社）（陽性検体）を、可溶性ＧＰＣ３評価用検体として使用した。前処理液として、６．２５ｍＭ～６００ｍＭの２－（ジメチルアミノ）エタンチオール塩酸塩（ＤＥＡＥＴ）を含むリン酸緩衝液（１０ｍＭリン酸緩衝液，６．２５～６００ｍＭ ＤＥＡＥＴ，ｐＨ６．０）を用いた。コントロール液として、１０ｍＭリン酸緩衝液（ｐＨ６．０）を用いた。陰性検体、陽性検体および緩衝液サンプル（１０ｍＭリン酸緩衝液）を、それぞれ、前処理液と体積比１：１で混合し、３７℃で６．５分間反応させた（前処理あり）。同様に、陰性検体、陽性検体および緩衝液サンプルを、それぞれ、コントロール液と混合し、反応させた（前処理なし）。 Example 1: Pretreatment of a sample with a reducing agent DEAET in the measurement of soluble GPC3 A serum sample derived from a healthy person (negative sample) and a serum sample derived from a liver cancer patient positive for α-fetoprotein (AFP) (Trina) (Positive sample) was used as a sample for evaluation of soluble GPC3. As the pretreatment solution, a phosphate buffer solution (10 mM phosphate buffer solution, 6.25-600 mM DEAET, pH 6.0) containing 6.25 mM to 600 mM 2- (dimethylamino) ethanethiol hydrochloride (DEAET) was used. board. As a control solution, a 10 mM phosphate buffer solution (pH 6.0) was used. Negative sample, positive sample and buffer sample (10 mM phosphate buffer) were mixed with the pretreatment solution at a volume ratio of 1: 1 and reacted at 37 ° C. for 6.5 minutes (with pretreatment). Similarly, the negative sample, the positive sample and the buffer solution sample were mixed with the control solution and reacted (without pretreatment).
各検体について、参考例４に記載の測定方法により可溶性ＧＰＣ３を測定した。各検体のカウントを表１に示す。また、カウントから下記計算式により算出された（１）低下率（％）、（２）陽性と陰性のカウント差（％）、および（３）マトリックス差（％）を表１に示す。
For each sample, soluble GPC3 was measured by the measuring method described in Reference Example 4. The count of each sample is shown in Table 1. Table 1 shows (1) reduction rate (%), (2) positive and negative count difference (%), and (3) matrix difference (%) calculated from the counts by the following formula.
計算式
（１）低下率（％）＝１００－（各ＤＥＡＥＴ濃度の「前処理あり」のカウント／「前処理なし」のカウント）×１００
（２）陽性と陰性のカウント差（％）＝（陽性検体のカウント平均値）／（陰性検体のカウント平均値）×１００
（３）マトリックス差（％）＝（緩衝液サンプルのカウント）／（陰性検体のカウント平均値）×１００ Calculation formula (1) Decrease rate (%) = 100- (count of "pretreatment" / count of "pretreatment" of each DEAET concentration) x 100
(2) Positive and negative count difference (%) = (average count of positive samples) / (average count of negative samples) x 100
(3) Matrix difference (%) = (count of buffer sample) / (average count of negative samples) x 100
（１）低下率（％）＝１００－（各ＤＥＡＥＴ濃度の「前処理あり」のカウント／「前処理なし」のカウント）×１００
（２）陽性と陰性のカウント差（％）＝（陽性検体のカウント平均値）／（陰性検体のカウント平均値）×１００
（３）マトリックス差（％）＝（緩衝液サンプルのカウント）／（陰性検体のカウント平均値）×１００ Calculation formula (1) Decrease rate (%) = 100- (count of "pretreatment" / count of "pretreatment" of each DEAET concentration) x 100
(2) Positive and negative count difference (%) = (average count of positive samples) / (average count of negative samples) x 100
(3) Matrix difference (%) = (count of buffer sample) / (average count of negative samples) x 100
低下率（％）は、個別検体における前処理の影響を評価するための指標である。陰性検体の低下率が大きいほど、また、陽性検体の低下率が小さいほど、陰性検体と陽性検体の測定値の差が広がり、両者をより区別可能となるため、高い診断感度を実現できる。したがって、陰性検体の低下率が大きいほど、また、陽性検体の低下率が小さいほど、可溶性ＧＰＣ３の測定系の前処理条件として有用である。
The rate of decrease (%) is an index for evaluating the effect of pretreatment on individual samples. The larger the decrease rate of the negative sample and the smaller the decrease rate of the positive sample, the wider the difference between the measured values of the negative sample and the positive sample, and the more distinguishable between the two, so that high diagnostic sensitivity can be realized. Therefore, the larger the reduction rate of the negative sample and the smaller the reduction rate of the positive sample, the more useful it is as a pretreatment condition for the measurement system of soluble GPC3.
陽性と陰性のカウント差（％）は、前処理による陽性検体と陰性検体のカウントの差を評価するための指標である。陽性と陰性のカウント差（％）が大きいほど、高い診断感度を実現できるため、可溶性ＧＰＣ３の測定系に有用である。
The positive and negative count difference (%) is an index for evaluating the difference between the counts of the positive sample and the negative sample due to the pretreatment. The larger the positive and negative count difference (%), the higher the diagnostic sensitivity can be realized, which is useful for the measurement system of soluble GPC3.
マトリックス差（％）は、緩衝液サンプルと陰性検体のカウントの差を評価するための指標である。検体を用いたイムノアッセイでは、検体に含まれる物質によって免疫反応が影響することがある（マトリックス効果）。マトリックス差（％）が１００％に近いほど、マトリックス効果の低減を実現できる。低減したマトリックス効果は、検体種に依存しない真値の出力を達成できるため、可溶性ＧＰＣ３の測定系に有用である。
The matrix difference (%) is an index for evaluating the difference in count between the buffer solution sample and the negative sample. In immunoassays using specimens, the substance contained in the specimen may affect the immune response (matrix effect). The closer the matrix difference (%) is to 100%, the more the matrix effect can be reduced. The reduced matrix effect is useful for the measurement system of soluble GPC3 because it can achieve a true value output independent of the sample species.
表１に示すように、陰性検体をＤＥＡＥＴで前処理した場合、検討した全ての濃度（６．２５ｍＭ～６００ｍＭ）において陰性検体のカウントは低下し、その低下率は非常に大きかった。一方、陽性検体をＤＥＡＥＴで前処理した場合、陽性検体のカウントは低下しないか、または低下しても陰性検体に比べてその低下率は小さかった。特に、ＤＥＡＥＴを５０ｍＭ～４００ｍＭの濃度で添加した場合、陽性と陰性のカウント差（％）は、非常に大きかった。このことは、還元剤ＤＥＡＥＴによる検体の前処理が可溶性ＧＰＣ３の測定における陽性検体と陰性検体との判別精度を向上できることを示す。また、ＤＥＡＥＴで陰性検体を前処理した場合、マトリックス差が１００％に近づき、マトリックス効果が低減された。したがって、還元剤ＤＥＡＥＴによる検体の前処理は、可溶性ＧＰＣ３の測定に有用であることが確認された。
As shown in Table 1, when the negative sample was pretreated with DEAET, the count of the negative sample decreased at all the concentrations examined (6.25 mM to 600 mM), and the decrease rate was very large. On the other hand, when the positive sample was pretreated with DEAET, the count of the positive sample did not decrease, or even if it decreased, the decrease rate was smaller than that of the negative sample. In particular, when DEAET was added at a concentration of 50 mM to 400 mM, the positive and negative count difference (%) was very large. This indicates that pretreatment of the sample with the reducing agent DEAET can improve the accuracy of discrimination between the positive sample and the negative sample in the measurement of soluble GPC3. Moreover, when the negative sample was pretreated with DEAET, the matrix difference approached 100%, and the matrix effect was reduced. Therefore, it was confirmed that the pretreatment of the sample with the reducing agent DEAET is useful for the measurement of soluble GPC3.
実施例２：可溶性ＧＰＣ３の測定における還元剤２ＭＥＡによる検体の前処理
前処理液として、６．２５ｍＭ～１００ｍＭの２－メルカプトエチルアミン塩酸塩（２ＭＥＡ）を含むリン酸緩衝液（１０ｍＭリン酸緩衝液，６．２５～１００ｍＭ ２ＭＥＡ，ｐＨ６．０）を用いた。コントロール液として、１０ｍＭリン酸緩衝液（ｐＨ６．０）を用いた。陰性検体、陽性検体および緩衝液サンプルを、それぞれ、前処理液と体積比１：１で混合し、３７℃で６．５分間反応させた（前処理あり）。同様に、陰性検体、陽性検体および緩衝液サンプルを、それぞれ、コントロール液と混合し、反応させた（前処理なし）。 Example 2: Pretreatment of Specimen with Reducing Agent 2MEA in Measurement of Soluble GPC3 Phosphate Buffer Solution (10 mM Phosphate Buffer Solution, 10 mM Phosphate Buffer Solution,) containing 6.25 mM to 100 mM 2-mercaptoethylamine hydrochloride (2MEA) as a pretreatment solution. 6.25 to 100 mM 2MEA, pH 6.0) was used. As a control solution, a 10 mM phosphate buffer solution (pH 6.0) was used. Negative samples, positive samples and buffer samples were mixed with the pretreatment solution at a volume ratio of 1: 1 and reacted at 37 ° C. for 6.5 minutes (with pretreatment). Similarly, the negative sample, the positive sample and the buffer solution sample were mixed with the control solution and reacted (without pretreatment).
前処理液として、６．２５ｍＭ～１００ｍＭの２－メルカプトエチルアミン塩酸塩（２ＭＥＡ）を含むリン酸緩衝液（１０ｍＭリン酸緩衝液，６．２５～１００ｍＭ ２ＭＥＡ，ｐＨ６．０）を用いた。コントロール液として、１０ｍＭリン酸緩衝液（ｐＨ６．０）を用いた。陰性検体、陽性検体および緩衝液サンプルを、それぞれ、前処理液と体積比１：１で混合し、３７℃で６．５分間反応させた（前処理あり）。同様に、陰性検体、陽性検体および緩衝液サンプルを、それぞれ、コントロール液と混合し、反応させた（前処理なし）。 Example 2: Pretreatment of Specimen with Reducing Agent 2MEA in Measurement of Soluble GPC3 Phosphate Buffer Solution (10 mM Phosphate Buffer Solution, 10 mM Phosphate Buffer Solution,) containing 6.25 mM to 100 mM 2-mercaptoethylamine hydrochloride (2MEA) as a pretreatment solution. 6.25 to 100 mM 2MEA, pH 6.0) was used. As a control solution, a 10 mM phosphate buffer solution (pH 6.0) was used. Negative samples, positive samples and buffer samples were mixed with the pretreatment solution at a volume ratio of 1: 1 and reacted at 37 ° C. for 6.5 minutes (with pretreatment). Similarly, the negative sample, the positive sample and the buffer solution sample were mixed with the control solution and reacted (without pretreatment).
各検体について、参考例４に記載の測定方法により可溶性ＧＰＣ３を測定した。各検体のカウントを表２に示す。また、実施例１と同様に、低下率（％）、陽性と陰性のカウント差（％）、およびマトリックス差（％）を算出した。
For each sample, soluble GPC3 was measured by the measuring method described in Reference Example 4. The count of each sample is shown in Table 2. Further, in the same manner as in Example 1, the reduction rate (%), the positive and negative count difference (%), and the matrix difference (%) were calculated.
表２に示すように、陰性検体を２ＭＥＡで前処理した場合、検討した全ての濃度（６．２５ｍＭ～１００ｍＭ）において陰性検体のカウントは低下し、その低下率は非常に大きかった。一方、陽性検体を２ＭＥＡで前処理した場合、陽性検体のカウントは低下しないか、または低下しても陰性検体に比べてその低下率は小さかった。このことは、還元剤２ＭＥＡによる検体の前処理が可溶性ＧＰＣ３の測定における陽性検体と陰性検体との判別精度を向上できることを示す。また、２ＭＥＡで陰性検体を前処理した場合、マトリックス差が１００％に近づき、マトリックス効果が低減された。したがって、還元剤２ＭＥＡによる検体の前処理は、可溶性ＧＰＣ３の測定に有用であることが確認された。
As shown in Table 2, when the negative sample was pretreated with 2MEA, the count of the negative sample decreased at all the concentrations examined (6.25 mM to 100 mM), and the decrease rate was very large. On the other hand, when the positive sample was pretreated with 2MEA, the count of the positive sample did not decrease, or even if it decreased, the decrease rate was smaller than that of the negative sample. This indicates that pretreatment of the sample with the reducing agent 2MEA can improve the accuracy of discrimination between the positive sample and the negative sample in the measurement of soluble GPC3. Further, when the negative sample was pretreated with 2MEA, the matrix difference approached 100%, and the matrix effect was reduced. Therefore, it was confirmed that the pretreatment of the sample with the reducing agent 2MEA is useful for the measurement of soluble GPC3.
実施例３：可溶性ＧＰＣ３の測定における還元剤ＴＣＥＰによる検体の前処理
前処理液として、６．２５ｍＭ～１００ｍＭのトリス（２－カルボキシエチル）ホスフィン塩酸塩（ＴＣＥＰ）を含むリン酸緩衝液（１０ｍＭリン酸緩衝液，６．２５～1００ｍＭ ＴＣＥＰ，ｐＨ６．０）を用いた。コントロール液として、１０ｍＭリン酸緩衝液（ｐＨ６．０）を用いた。陰性検体、陽性検体および緩衝液サンプルを、それぞれ、前処理液と体積比１：１で混合し、３７℃で６．５分間反応させた（前処理あり）。同様に、陰性検体、陽性検体および緩衝液サンプルを、それぞれ、コントロール液と混合し、反応させた（前処理なし）。 Example 3: Pretreatment of Samples with Reducing Agent TCEP in Measurement of Soluble GPC3 Phosphate Buffer (10 mM phosphorus) containing 6.25 mM-100 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) as a pretreatment solution. Acid buffer, 6.25-100 mM TCEP, pH 6.0) was used. As a control solution, a 10 mM phosphate buffer solution (pH 6.0) was used. Negative samples, positive samples and buffer samples were mixed with the pretreatment solution at a volume ratio of 1: 1 and reacted at 37 ° C. for 6.5 minutes (with pretreatment). Similarly, the negative sample, the positive sample and the buffer solution sample were mixed with the control solution and reacted (without pretreatment).
前処理液として、６．２５ｍＭ～１００ｍＭのトリス（２－カルボキシエチル）ホスフィン塩酸塩（ＴＣＥＰ）を含むリン酸緩衝液（１０ｍＭリン酸緩衝液，６．２５～1００ｍＭ ＴＣＥＰ，ｐＨ６．０）を用いた。コントロール液として、１０ｍＭリン酸緩衝液（ｐＨ６．０）を用いた。陰性検体、陽性検体および緩衝液サンプルを、それぞれ、前処理液と体積比１：１で混合し、３７℃で６．５分間反応させた（前処理あり）。同様に、陰性検体、陽性検体および緩衝液サンプルを、それぞれ、コントロール液と混合し、反応させた（前処理なし）。 Example 3: Pretreatment of Samples with Reducing Agent TCEP in Measurement of Soluble GPC3 Phosphate Buffer (10 mM phosphorus) containing 6.25 mM-100 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) as a pretreatment solution. Acid buffer, 6.25-100 mM TCEP, pH 6.0) was used. As a control solution, a 10 mM phosphate buffer solution (pH 6.0) was used. Negative samples, positive samples and buffer samples were mixed with the pretreatment solution at a volume ratio of 1: 1 and reacted at 37 ° C. for 6.5 minutes (with pretreatment). Similarly, the negative sample, the positive sample and the buffer solution sample were mixed with the control solution and reacted (without pretreatment).
各検体について、参考例４に記載の測定方法により可溶性ＧＰＣ３を測定した。各検体のカウントを表３に示す。また、実施例１と同様に、低下率（％）、陽性と陰性のカウント差（％）、およびマトリックス差（％）を算出した。
For each sample, soluble GPC3 was measured by the measuring method described in Reference Example 4. The count of each sample is shown in Table 3. Further, in the same manner as in Example 1, the reduction rate (%), the positive and negative count difference (%), and the matrix difference (%) were calculated.
表３に示すように、陰性検体をＴＣＥＰで前処理した場合、検討した全ての濃度（６．２５ｍＭ～１００ｍＭ）において陰性検体のカウントは低下し、その低下率は非常に大きかった。一方、陽性検体をＴＣＥＰで前処理した場合、陽性検体のカウントは低下しないか、または低下しても陰性検体に比べてその低下率は小さかった。このことは、還元剤ＴＣＥＰによる検体の前処理が可溶性ＧＰＣ３の測定における陽性検体と陰性検体との判別精度を向上できることを示す。また、ＴＣＥＰで陰性検体を前処理した場合、マトリックス差が１００％に近づき、マトリックス効果が低減された。したがって、還元剤ＴＣＥＰによる検体の前処理は、可溶性ＧＰＣ３の測定に有用であることが確認された。
As shown in Table 3, when negative samples were pretreated with TCEP, the count of negative samples decreased at all the concentrations examined (6.25 mM to 100 mM), and the rate of decrease was very large. On the other hand, when positive samples were pretreated with TCEP, the count of positive samples did not decrease, or even if it decreased, the rate of decrease was smaller than that of negative samples. This indicates that pretreatment of the sample with the reducing agent TCEP can improve the accuracy of discrimination between the positive sample and the negative sample in the measurement of soluble GPC3. In addition, when a negative sample was pretreated with TCEP, the matrix difference approached 100% and the matrix effect was reduced. Therefore, it was confirmed that pretreatment of the sample with the reducing agent TCEP is useful for the measurement of soluble GPC3.
（実施例１～３のまとめ）
実施例１～３の結果を考慮すると、還元剤による検体の前処理は、還元剤の種類および構造にかかわらず、可溶性ＧＰＣ３の測定における陽性検体と陰性検体との判別精度を向上できると考えられる。また、還元剤による陰性検体の前処理は、マトリックス効果の低減により、検体種に依存しない真値の出力を実現できると考えられる。したがって、還元剤による検体の前処理は、可溶性ＧＰＣ３の測定に有用である。 (Summary of Examples 1 to 3)
Considering the results of Examples 1 to 3, it is considered that the pretreatment of the sample with the reducing agent can improve the discrimination accuracy between the positive sample and the negative sample in the measurement of soluble GPC3 regardless of the type and structure of the reducing agent. .. Further, it is considered that the pretreatment of the negative sample with the reducing agent can realize the output of the true value independent of the sample species by reducing the matrix effect. Therefore, pretreatment of the sample with a reducing agent is useful for the measurement of soluble GPC3.
実施例１～３の結果を考慮すると、還元剤による検体の前処理は、還元剤の種類および構造にかかわらず、可溶性ＧＰＣ３の測定における陽性検体と陰性検体との判別精度を向上できると考えられる。また、還元剤による陰性検体の前処理は、マトリックス効果の低減により、検体種に依存しない真値の出力を実現できると考えられる。したがって、還元剤による検体の前処理は、可溶性ＧＰＣ３の測定に有用である。 (Summary of Examples 1 to 3)
Considering the results of Examples 1 to 3, it is considered that the pretreatment of the sample with the reducing agent can improve the discrimination accuracy between the positive sample and the negative sample in the measurement of soluble GPC3 regardless of the type and structure of the reducing agent. .. Further, it is considered that the pretreatment of the negative sample with the reducing agent can realize the output of the true value independent of the sample species by reducing the matrix effect. Therefore, pretreatment of the sample with a reducing agent is useful for the measurement of soluble GPC3.
実施例４：可溶性ＧＰＣ３の測定における還元剤と界面活性剤の組合せによる検体の前処理
前処理液として、表４Ａに示す試験例２～１１のリン酸緩衝液（ｐＨ６．０）を用いた。試験例２～１１のリン酸緩衝液は、還元剤としてＤＥＡＥＴ、および界面活性剤として３－［（３－コールアミドプロピル）ジメチルアンモニオ］－１－プロパンスルホネート（ＣＨＡＰＳ）、またはポリオキシエチレンソルビタンモノラウラート（Ｔｗｅｅｎ２０）を含んでいた。コントロール液として、表４Ａに示す試験例１の１０ｍＭリン酸緩衝液（ｐＨ６．０）を用いた。陰性検体、陽性検体および緩衝液サンプルを、それぞれ、前処理液と体積比１：１で混合し、３７℃で６．５分間反応させた（前処理あり）。同様に、陰性検体、陽性検体および緩衝液サンプルを、それぞれ、コントロール液と混合し、反応させた（前処理なし）。 Example 4: Pretreatment of a sample with a combination of a reducing agent and a surfactant in the measurement of soluble GPC3 As the pretreatment liquid, the phosphate buffer solution (pH 6.0) of Test Examples 2 to 11 shown in Table 4A was used. The phosphate buffers of Test Examples 2 to 11 are DEAET as a reducing agent and 3-[(3-colamidepropyl) dimethylammonio] -1-propanesulfonate (CHAPS) as a surfactant, or polyoxyethylene sorbitan. It contained monolaurate (Tween 20). As the control solution, the 10 mM phosphate buffer solution (pH 6.0) of Test Example 1 shown in Table 4A was used. Negative samples, positive samples and buffer samples were mixed with the pretreatment solution at a volume ratio of 1: 1 and reacted at 37 ° C. for 6.5 minutes (with pretreatment). Similarly, the negative sample, the positive sample and the buffer solution sample were mixed with the control solution and reacted (without pretreatment).
前処理液として、表４Ａに示す試験例２～１１のリン酸緩衝液（ｐＨ６．０）を用いた。試験例２～１１のリン酸緩衝液は、還元剤としてＤＥＡＥＴ、および界面活性剤として３－［（３－コールアミドプロピル）ジメチルアンモニオ］－１－プロパンスルホネート（ＣＨＡＰＳ）、またはポリオキシエチレンソルビタンモノラウラート（Ｔｗｅｅｎ２０）を含んでいた。コントロール液として、表４Ａに示す試験例１の１０ｍＭリン酸緩衝液（ｐＨ６．０）を用いた。陰性検体、陽性検体および緩衝液サンプルを、それぞれ、前処理液と体積比１：１で混合し、３７℃で６．５分間反応させた（前処理あり）。同様に、陰性検体、陽性検体および緩衝液サンプルを、それぞれ、コントロール液と混合し、反応させた（前処理なし）。 Example 4: Pretreatment of a sample with a combination of a reducing agent and a surfactant in the measurement of soluble GPC3 As the pretreatment liquid, the phosphate buffer solution (pH 6.0) of Test Examples 2 to 11 shown in Table 4A was used. The phosphate buffers of Test Examples 2 to 11 are DEAET as a reducing agent and 3-[(3-colamidepropyl) dimethylammonio] -1-propanesulfonate (CHAPS) as a surfactant, or polyoxyethylene sorbitan. It contained monolaurate (Tween 20). As the control solution, the 10 mM phosphate buffer solution (pH 6.0) of Test Example 1 shown in Table 4A was used. Negative samples, positive samples and buffer samples were mixed with the pretreatment solution at a volume ratio of 1: 1 and reacted at 37 ° C. for 6.5 minutes (with pretreatment). Similarly, the negative sample, the positive sample and the buffer solution sample were mixed with the control solution and reacted (without pretreatment).
各検体について、参考例４に記載の測定方法により可溶性ＧＰＣ３を測定した。各検体のカウントを表４Ｂに示す。また、実施例１と同様に、低下率（％）、陽性と陰性のカウント差（％）、およびマトリックス差（％）を算出した。
For each sample, soluble GPC3 was measured by the measuring method described in Reference Example 4. The count of each sample is shown in Table 4B. Further, in the same manner as in Example 1, the reduction rate (%), the positive and negative count difference (%), and the matrix difference (%) were calculated.
表４に示すように、陰性検体を還元剤と界面活性剤の組合せで前処理した場合、検討した全ての界面活性剤濃度（各０．１～１０重量％）において陰性検体のカウントは低下し、その低下率は非常に大きかった。一方、陽性検体を還元剤と界面活性剤の組合せで前処理した場合、陽性検体のカウントの低下率は陰性検体に比べて小さかった。特に、界面活性剤を所定の濃度で添加した場合（０．１～２．５重量％ ＣＨＡＰＳ、および０．１～１０重量％ Ｔｗｅｅｎ２０）、陽性と陰性のカウント差（％）は、非常に大きかった。また、還元剤と界面活性剤の組合せで陰性検体を前処理した場合、マトリックス差がより１００％に近づき、マトリックス効果をさらに低減された。このことは、還元剤と界面活性剤の組合せによる検体の前処理が可溶性ＧＰＣ３の測定における陽性検体と陰性検体との判別精度を向上できること、および検体のマトリックス効果を低減できることを示す。したがって、還元剤と界面活性剤の組合せによる検体の前処理は、可溶性ＧＰＣ３の測定に有用であることが確認された。
As shown in Table 4, when the negative sample was pretreated with a combination of a reducing agent and a surfactant, the count of the negative sample decreased at all the surfactant concentrations examined (0.1 to 10% by weight each). , The rate of decrease was very large. On the other hand, when the positive sample was pretreated with a combination of a reducing agent and a surfactant, the rate of decrease in the count of the positive sample was smaller than that of the negative sample. In particular, when the surfactant is added at a predetermined concentration (0.1 to 2.5% by weight CHAPS and 0.1 to 10% by weight Tween 20), the positive and negative count difference (%) is very large. rice field. Further, when the negative sample was pretreated with the combination of the reducing agent and the surfactant, the matrix difference became closer to 100%, and the matrix effect was further reduced. This indicates that the pretreatment of the sample with the combination of the reducing agent and the surfactant can improve the accuracy of discriminating between the positive sample and the negative sample in the measurement of soluble GPC3, and can reduce the matrix effect of the sample. Therefore, it was confirmed that the pretreatment of the sample with the combination of the reducing agent and the surfactant is useful for the measurement of soluble GPC3.
Claims (15)
- 可溶性ＧＰＣ３含有検体を還元剤と混合することを含む、可溶性ＧＰＣ３のイムノアッセイにおける可溶性ＧＰＣ３含有検体の処理方法。 A method for treating a soluble GPC3-containing sample in an immunoassay for soluble GPC3, which comprises mixing the soluble GPC3-containing sample with a reducing agent.
- 還元剤が、０．０５～１，０００ｍＭの終濃度で用いられる、請求項１記載の方法。 The method according to claim 1, wherein the reducing agent is used at a final concentration of 0.05 to 1,000 mM.
- 可溶性ＧＰＣ３含有検体を界面活性剤とさらに混合することを含む、請求項１または２記載の方法。 The method according to claim 1 or 2, which comprises further mixing a soluble GPC3-containing sample with a surfactant.
- 界面活性剤が、０．００５～１０重量％の終濃度で用いられる、請求項３記載の方法。 The method according to claim 3, wherein the surfactant is used at a final concentration of 0.005 to 10% by weight.
- イムノアッセイが、可溶性ＧＰＣ３中の異なる２種以上のエピトープに対する異なる２種以上の抗体を用いるサンドイッチイムノアッセイである、請求項１～４のいずれか一項記載の方法。 The method according to any one of claims 1 to 4, wherein the immunoassay is a sandwich immunoassay using two or more different antibodies against two or more different epitopes in soluble GPC3.
- 可溶性ＧＰＣ３含有検体が血液検体である、請求項１～５のいずれか一項記載の方法。 The method according to any one of claims 1 to 5, wherein the soluble GPC3-containing sample is a blood sample.
- 可溶性ＧＰＣ３含有検体が、癌に罹患している被験体から得られる、請求項１～６のいずれか一項記載の方法。 The method according to any one of claims 1 to 6, wherein the soluble GPC3-containing sample is obtained from a subject suffering from cancer.
- 癌が肝臓癌である、請求項７記載の方法。 The method according to claim 7, wherein the cancer is liver cancer.
- 下記（ａ）および（ｂ）を含む、可溶性ＧＰＣ３のイムノアッセイ方法：
（ａ）可溶性ＧＰＣ３含有検体を還元剤と混合すること；および
（ｂ）（ａ）で得られた混合液中の可溶性ＧＰＣ３量を、可溶性ＧＰＣ３に対する１種以上の抗体を用いて測定すること。 Immunoassay method for soluble GPC3, including the following (a) and (b):
(A) Mixing a soluble GPC3-containing sample with a reducing agent; and (b) measuring the amount of soluble GPC3 in the mixture obtained in (a) using one or more antibodies against soluble GPC3. - 可溶性ＧＰＣ３含有検体を界面活性剤とさらに混合することを含む、請求項９記載の方法。 The method according to claim 9, wherein the soluble GPC3-containing sample is further mixed with a surfactant.
- 測定が、可溶性ＧＰＣ３中の異なる２種以上のエピトープに対する異なる２種以上の抗体を用いるサンドイッチイムノアッセイにより行われる、請求項９または１０記載の方法。 The method of claim 9 or 10, wherein the measurement is performed by a sandwich immunoassay using two or more different antibodies against two or more different epitopes in soluble GPC3.
- 下記（ａ）および（ｂ）を含む、可溶性ＧＰＣ３のイムノアッセイ試薬：
（ａ）還元剤；および
（ｂ）可溶性ＧＰＣ３に対する１種以上の抗体。 Soluble GPC3 Immunoassay Reagents Containing (a) and (b):
(A) Reducing agent; and (b) One or more antibodies against soluble GPC3. - さらに（ｃ）界面活性剤を含む、請求項１２記載のイムノアッセイ試薬。 The immunoassay reagent according to claim 12, further comprising (c) a surfactant.
- 可溶性ＧＰＣ３に対する１種以上の抗体が、可溶性ＧＰＣ３中の異なる２種以上のエピトープに対する異なる２種以上の抗体であり、かつ、前記イムノアッセイ試薬がサンドイッチイムノアッセイ用である、請求項１２または１３記載のイムノアッセイ試薬。 The immunoassay according to claim 12 or 13, wherein the one or more antibodies against soluble GPC3 are two or more different antibodies against two or more different epitopes in soluble GPC3, and the immunoassay reagent is for sandwich immunoassay. reagent.
- 前記試薬が癌の診断用である、請求項１２～１４のいずれか一項記載のイムノアッセイ試薬。 The immunoassay reagent according to any one of claims 12 to 14, wherein the reagent is for diagnosing cancer.
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| CN116802495A (en) | 2023-09-22 |
| EP4279504A1 (en) | 2023-11-22 |
| JPWO2022154119A1 (en) | 2022-07-21 |
| US20240094210A1 (en) | 2024-03-21 |
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